Tissue-specific nuclear repatterning in plant cells

Tissue-specific nuclear repatternings, consisting of changes in the number and size of the chromocenters, were observed by analyzing, in Feulgen squashes and sections, different tissues of several plant species, particularly of Ionopsidium savianum. Nuclear repatternings occur mainly near the base of the meristems. They are due to associations of chromosomes at their heterochromatic regions. This was confirmed by the results of cytophotometric measurements, showing the same contents of both Feulgen/DNA and heterochromatin in nuclei with a different number of chromocenters. These data also showed that chromosome association does not occur in endoreduplicating or endoreduplicated cells. Autoradiographic results after [3H]thymidine treatments indicated that DNA synthesis does not occur in nuclei with extensive chromosome association. A highly significant, positive correlation was found between the number of chromocenters in each nucleus and the amount of RNA synthesis as indicated by [3H]uridine incorporation. It is suggested that chromosome association plays some role in the regulation of the functional activity of the nucleus and in tissue differentiation.     

Localization of late replicating DNA and nuclear membrane

We investigated whether the specific localization of DNA replicator sites at the nuclear membrane at the end of the S phase, found in dividing animal cells and in the plantHaplopappus gracilis, applied also to other plants. We found that nuclei labelled at the nuclear periphery were observed in plants where the chromocenters are localized at the nuclear membrane. In other plants, where the chromocenters are scattered throughout the nucleus, a different pattern of labelling is observed where the silver grains are restricted to a number of distinct sites, distributed in a fashion similar to that of the chromocenters. We believe that these nuclei were replicating the chromocentric heterochromatin and so were therefore at the end of the S phase. The specific patterns of the distribution of DNA replicator sites at the end of the S phase make it possible to distinguish nuclei which are in the late S phase and thus define a specific stage of the S phase, during which only heterochromatin replication occurs.     

Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. II. Mechanisms of polyploidization

Peculiarities of the structure of interphase nuclei, mitotic activity, and Ki-67 protein intranuclear immunolocalization were studied to elucidate mechanisms of genome multiplication in proliferative and differentiating invasive extravillous trophoblast cells in the human placenta. The presence of numerous chromocenters was shown to be a characteristic feature of proliferative cell nuclei of both villous and extravillous trophoblast. At the beginning of extravillous trophoblast cell differentiation, i.e. in the proximal part of cell columns, some amount of cells with large nuclei containing enlarged chromocenters were found. DNA content was measured simultaneously with counting the number of chromocenters in similarly looking nuclei of squash preparations of placental villi. The increase in the ploidy level up to 4c-8c, accompanied by a slight increase in the number of chromocenters being not proportional to the ploidy level and not exceeding the diploid number of chromosomes of the human genome, was demonstrated. This suggests that genome multiplication of extravillous trophoblast cells may be accomplished by endoreduplication. In addition, pictures of endomitosis were seen at early steps of differentiation of EVT cells. The lack of polyploid mitotic figures or any obvious polyploidizing or restitutional mitoses suggests that these are not of considerable importance in genome multiplication of human EVT cells. However, the prevalence of metaphases at the boundary of the distal part of cell columns suggests that restitutional mitoses may be involved, even partly, in human trophoblast cell polyploidization. At later steps of differentiation, i.e. in the distal part of cell columns, the nuclear structure obviously changes, with a uniform "network" chromatin arrangement prevailing, whereas numerous chromocenters and features of endomitosis are no longer seen. The pattern of Ki-67 protein immunolocalization is also changing along the invasive pathway. In the proliferating stem cells and trophoblast cells of the proximal part of cell columns, Ki-67 was localized in the karyoplasm, chromocenters and numerous small nucleoli, whereas in the distal part of cell columns this protein was detected predominantly in 1-2 large nucleoli. The comparative analysis of the literature data on Ki-67 localization at different stages of cell cycle provided another evidence that EVT cells in the course of invasion may switch to the endoreduplication cycle. In agreement with the relevant report on rodent placentation, our present data suggest that acquirement of an invasive phenotype of EVT cells is accompanied by switching from mitotic division to endoreduplication cycle.     

Isolation of the Chromocenter Fraction from Mouse Liver Nuclei

A new method for isolation of the constitutive heterochromatin (chromocenters) from interphase nuclei of mouse liver has been developed. This method allows separation of chromocenters of different size. Chromocenter fractions are essentially free of nucleoli and other contaminants. In contrast to nuclei and nucleoli, the chromocenter fraction is characterized by simpler protein composition, this fraction having a reduced number of proteins (especially high molecular weight proteins). Chromocenters contain all histone fractions; however, the relative proportion of histone H1 is lower and histone H3 is higher than in the total nuclear chromatin. The amount of non-histone proteins of 51, 63, 73, and 180 kD is higher in the chromocenter fraction than in nuclei and nucleoli. The use of immunocytochemistry and immunoblotting methods revealed the presence of the specific kinetochore component, CENP A protein. This suggests tight association of some molecular kinetochore components with chromocenters in the interphase.     

Condensed chromatin and its underreplication during root differentiation in leguminosae

Interphase nuclear structure was studied in 15 leguminous species. Eleven species showed chromocentric interphase nuclei while the remaining 4 had reticulate nuclei. The number of chromocenters appeared to be dependent on the number of chromosomes (2n). The total proportion of condensed chromatin as determined by planimetry was found to vary from 11–24% in chromocentric nuclei and 29–62% in reticulate nuclei. The condensed chromatin amount showed a direct correlation with the nuclear DNA content (2C). Though the interphase nuclear structure remained same in differentiated cells, the amount of condensed chromatin was considerably less than that in the meristematic cells, indicating underreplication of heterochromatin during differentiation. HCl-Giemsa method seems to be the simplest method for detection of underreplication in plants.1. NCL Communication No. 35942. To whom all the correspondence should be addressed     

The diagnosis of the sex chromosomes in human tissues

Summary The nuclei in the skin, and in the neutrophils, have been studied in men and women, in relation to a diagnosis of the sex chromosomes in non-dividing nuclei.It has been shown in the skin, that the appearance of chromocenters, which are presumably formed by the X and Y, can vary according to metabolic conditions, but that a determination of the percent of nuclei with different numbers of chromocenters and nucleoli in the young and old spinous cells, gives a characteristic distribution of nuclear types in each of the two sexes.Since such a determination includes cells with individual X and Y chromocenters, it should be possible to detect by this method not only cases that are XX or XY, but also cases with abnormal sex chromosome constitutions.     

A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis

A method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone. The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two-thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S-phase cells.     

大鼠衰老过程中脑细胞DNA与c－Ha－ras原癌基因的甲基化

用ＭｓｐⅠ／ＨｐａⅡ酶解电泳法和高效液相色谱（ＨＰＬＣ）两种方法进行比较,研究了不同年龄大鼠的肝、脑细胞基因组ＤＮＡ的甲基化程度。从酶解电泳图谱可观察到,肝、脑细胞基因组ＤＮＡ甲基化在青年鼠和老年鼠之间没有差异。但用具有高分辨率的高效液相色谱测量ＤＮＡ中５－ｍＣ的含量时发现,老年鼠脑细胞ＤＮＡ甲基化程度较大年鼠的下降６２％,而肝细胞ＤＮＡ甲基化程度在老年鼠与青年鼠之间并没有显著差异。这些结果提示：（１）用常规的酶解电泳法所分析的ＤＮＡ甲基化结果并不能反映整个基因组ＤＮＡ甲基化的水平。（２）衰老过程中,不同组织ＤＮＡ甲基化的改变存在差异,引起这种差异的原因可能与组织的增殖和分化程度有关。进一步分析脑细胞原癌基因ｃ－Ｈａ－ｒａｓ的甲基化水平,无论ＭｓｐⅠ酶切图谱,还是ＨｐａⅡ酶切图谱均可观察到分子大小为１９ｋｂ、７．５ｋｂ、１．３ｋｂ、０．９ｋｂ的四条阳性带,说明该基因未发生甲基化,且与年龄无关。     

Chromocenters of interphase nuclei in the mouse liver contain satellite DNA]

In isolated interphase mouse liver nuclei after hypotonic treatment only the chromocenters belonging to the pericentromeric heterochromatin remain in dense form while the main mass of a chromatin is completely decondensed. The centromeric nature of these chromocenters is demonstrated by their capability for C-banding and for hybridization with a satellite mouse DNA.     

Cytological studies on the organization of DNA in giant trophoblast nuclei of the mouse and the rat

Giant trophoblast nuclei of the mouse and the rat, known to contain hundreds, or even thousands, of times the haploid amount of DNA, have been studied by a number of cytological techniques. These nuclei appear in two morphological states:“reticulate,” in which large numbers of chromatin threads of uniform size intermingle throughout the nucleus, often radiating from clumps of heterochromatin adjacent to the nucleoli, and “homogeneous,” in which the chromatin is more evenly dispersed and individual threads are more difficult to distinguish. Intermediate morphologies are also observed. In neither case were structures resembling polytene chromosomes discernible. — Centromeric heterohromatin as revealed by the Giemsa BSG technique has been quantitatively analyzed in giant versus diploid trophoblast nuclei. Although the median number of chromocenters is slightly greater in giant than in diploid nuclei, the range is similar. In both cases, the chromocenter number is usually less than the diploid number of chromosome pairs, indicating the attraction between centromeres not only of homologous, but also of heterologous, chromosomes. By scanning microdensitometry, we have observed a constant ratio of chromocenter area: total nuclear area in giant cells. This ratio, which likely reflects the ratio of chromocenter volume: total nuclear volume, is in good agreement with that of satellite DNA: total DNA.     

Senescence vs. changes in nuclear DNA, protein and dry mass contents in leaf mesophyll of two species differing in occurrence of endomitotic polyploidy

DNA and Naphtol yellow S-staining (F-NYS) protein contents were measured cytophotometrically using the Feulgen method in the nuclei of the mesophyll from the basal and apical zone of young and old leaves in two perennial monocotyledonous species: Rhoeo discolor and Clivia miniata, differing in presence or absence of DNA endoreplication. Dry mass content was determined interferometrically using an uniform field with large image shearing method. It has been shown that nuclei with 2C DNA and below 2C DNA content dominate in old leaves. The decrease in dry mass content of nuclei correlated with the decrease in NYS protein content. Parallelly a significant increase in NYS protein and DNA contents observed in chromocenters Rhoeo discolor was proportional to the increase in their dry mass. The decrease in nuclear DNA content in mesophyll of old leaves in endoreplicating species was the same as in non-edoreplicating one, however the senescence was more intensive in endoreplicating species.     

Mitochondrial DNA sequences are present inside nuclear DNA in rat tissues and increase with age

Mitochondrial DNA (mtDNA) mutations increase with age. However, the number of cells with predominantly mutated mtDNA is small in old animals. Here a new hypothesis is proposed: mtDNA fragments may insert into nuclear DNA contributing to aging and related diseases by alterations in the nucleus. Real-time PCR quantification shows that sequences of cytochrome oxidase III and 16S rRNA from mtDNA are present in highly purified nuclei from liver and brain in young and old rats. The sequences of these insertions revealed that they contain single nucleotide polymorphisms identical to those present in mtDNA of the same animal. Interestingly, the amount of mitochondrial sequences in nuclear DNA increases with age in both tissues. In situ hybridization of mtDNA to nuclear DNA confirms the presence of mtDNA sequences inside nuclear DNA in rat hepatocytes. Bone marrow metaphase cells from both young and old rats show mtDNA at centromeric regions in 20 out of the 2n = 40 chromosomes. Consequently, mitochondria can be a major trigger of aging but the final target could also be the nucleus.     

Changes in the function of the nucleus and chromatin of human cells under the action of hormonal factors in vitro

The influence of hormones--adrenaline, noradrenaline, hydrocortisone on the electronegativity of cell nuclei (ENN) was shown by the method of intracellular microelectrophoresis. Changes in heterochromatin state were estimated on the basis of heterochromatin granules (chromocenters) quantity. Adrenaline and noradrenaline induced the decrease in ENN, the maximum effect was revealed with hormones concentration observed in human blood during physical training. The hormonal effect increased with gradual increase of the exposition time from 5 to 120 min. Hydrocortisone also decreased ENN. Adrenaline, and in some cases hydrocortisone, induced the significant increase of heterochromatin granule quantity. The correlation of this process with ENN decrease was shown only in the case of adrenaline. Obtained data are discussed in connection with regulation mechanisms of cell nucleus functional activity.     

Evidence for attachment of interphase chromatin to the nuclear matrix via matrix-bound nucleosomes

Chromatin structure has been studied in the sites of attachment to the nuclear matrix in interphase mouse liver and spleen nuclei. The patterns of fragmentation of the DNA belonging to these sites (0.3-2% of total DNA in spleen and liver, respectively) with staphylococcal nuclease and DNAase I were very close to those of usual nucleosomal chains. Moreover, the nuclear matrix preparations contained all five major histones, including H1, in almost stoichiometric amounts. The histone/DNA ratios for the matrix were also similar to those found in nuclei. These findings and the size of the matrix-protected DNA indicated that interphase chromatin was attached to the nuclear matrix via matrix-bound nucleosomes and, to a much lesser extent, oligonucleosomes up to 5-6 units long. Two-dimensional electrophoretic separation of the matrix-bound histones revealed that modifications of histone H1 and, probably, of other histones were distinguished from those in bulk chromatin. Study of binding of exogenously added labeled histone octamers or mononucleosomal size DNA to nuclear matrix excluded the possibility of their artifactual trapping during the isolation procedure.     

Distribution of myonuclei and microtubules in live muscle fibers of young, middle-aged, and old mice.

We have recently published a new technique for visualizing nuclei in living muscle fibers of intact animals, based on microinjection of labeled DNA into single myofibers, excluding satellite cells (Bruusgaard JC, Liestol K, Ekmark M, Kollstad K, and Gundersen K. J Physiol 551: 467-478, 2003). In the present study, we use this technique to study fiber segments of soleus and extensor digitorum longus (EDL) muscles from mice aged 2, 14, and 23 mo. As the animals maturing from 2 to 14 mo, they displayed an increase in size and number of nuclei. Soleus showed little change in nuclear domain size, whereas this increased by 88% in the EDL. For 14-mo-old animals, no significant correlation between fiber size and nuclear number was observed (R2=0.18, P=0.51) despite a fourfold variation in cytoplasmic volume. This suggests that size and nuclear number is uncoupled in middle-aged mice. When animals aged from 14 to 23 mo, EDL IIb, but not soleus, fibers atrophied by 41%. Both EDL and soleus displayed a reduction in number of nuclei: 20 and 16%, respectively. A positive correlation between number of nuclei and size was observed at 2 mo, and this reappeared in old mice. The atrophy in IIb fibers at old age was accompanied by a disturbance in the orderly positioning of nuclei that is so prominent in glycolytic fibers at younger age. In old animals, changes in nuclear shape and in the peri- and internuclear microtubule network were also observed. Thus changes in myonuclear number and distribution, perhaps related to alterations in the microtubular network, may underlie some of the adverse consequences of aging on skeletal muscle size and function.     

A simple fluorescence staining technique for the differentiation of human tissue transplanted into nude mice.

Human and mouse nuclei can be distinguished by differences in the constitutive heterochromatin when stained with quinacrine dihydrochloride. With the staining method described, mouse heterochromatin during interphase appears as brilliant fluorescent chromocenters. By replacing the commonly used aqueous buffer mounting medium with a xylene-diluted synthetic resin, the haziness of the nuclear fluorescence is eliminated thus allowing identification of the heterochromatin pattern in histological preparations. A requirement for the definite identification of cells of human or murine origin in the nude mouse is the knowledge that the heterochromatin arrangements changes according to the stage of differentiation of the cell of the position of a particular nucleus within the cell cycle.     

The metabolism of nicotinamide-adenine dinucleotide in various classes of rat liver nuclei.

1. The activities of NMN adenylyltransferase and an enzyme that synthesizes poly (ADP-ribose) from NAD were investigated in the various classes of rat liver nuclei fractionated by zonal centrifugation. 2. The highest specific activities of these two nuclear enzymes occur in different classes of nuclei. In very young and in mature rats it was shown that a correlation exists between DNA synthesis and NMN adenylyltransferase activity, but in rats of intermediate age this correlation is less evident. The highest activities of the enzyme that catalyses formation of poly (ADP-ribose) are in the nuclei involved in the synthesis of RNA. 3. The significance of these results in relation to NAD metabolism is discussed.     

The possible role of endogenous nucleases in the structural organization of chromatin]

Two fractions of rat liver nuclei with different buoyant density have been obtained. The electrophoretic analysis of the oligonucleosome patterns of DNA out of nuclei of these two fractions revealed different levels of activity in endonucleases. In case of inhibition during the extraction of activity in Ca, Mg-dependent endonucleases, the average size of high polymeric DNA is larger for nuclei with bigger buoyant density (fraction I) than for nuclei with smaller ones (fraction II). This finding is evidence of in situ existence of two pools of liver nuclei with different endogenic nuclease activities. In nuclear chromatin fraction I DNA is torsionally stressed; in fraction II it is relaxed that correlates with larger activity of endonucleases and smaller buoyant density of this fraction. A hypothesis on a possible role of endonucleases in chromatin structure organization has been put forward. According to this hypothesis a modulation of activity in nuclear endonucleases can determine different packaging and activity of chromatin from different pools of cellular nuclei.     

DNA metabolism during maturation of male germ cells. II. Amplification structures in the nuclei of rat germ and somatic cells

Amplification structures have been found in preparations of histone-depleted somatic (liver) and sex (spermatogonia, spermatocytes 1) rat cells. Multi-forked chromosomal (2-4 replicative forks originating from a single strand of DNA) and extrachromosomal circular amplification structures have been detected in the nuclei of sex cells. All the circular molecules of DNA detected belong, according to size, to the class of small nuclear polydispersed circular DNAs. Chromosomal amplification structures (eye-in-eye or several replicative forks originating from one DNA strand) have been only detected in the nuclei of somatic cells.     

Preparation of isolated cells by alkaline dissociation of formalin fixed tissue]

A procedure is described for preparation of isolated cells by treating formaldehyde fixed tissues with a 50%-solution of KOH. This results in complete yield of cells from a variety of organs (liver, kidney, heart, spleen, etc.). The alkali-treated cells entirely retain their morphological and tinctorial peculiarities. It was shown that preparations derived from alkali-treated tissues were useful for a series of quantitative cytological and cytochemical techniques: cytofluorometric estimation of nuclear DNA; interferometric determination of dry cell mass; autoradiographic studies of nuclear DNA synthesis; cell number counts; evaluation of cell distribution according to the number of their nuclei; estimation of the mitotic index.