Fluorometric identification of chromatin packing level laying out of the light microscopy resolution

Confocal microscopy permits to perform quantitative analysis of the fluorescent signals of the nuclei. We determined the level of DAPI and phosphorylated histone-H3 fluorescence, the volume of DAPI and phosphorylated histone-H3 fluorescence in normal (Hikone AW) and colchicine treated third instar Drosophila melanogaster wing imaginal disc mitotic cells. Our analysis permitted to indentify two unknown levels of chromatin package; one in the prometaphase and another at the end of metaphase. These levels disappeared in colchicine treated mitoses. We conclude that quantitative analysis of confocal images is able to detect the differences in chromatin package laying over the resolution of light microscopy.     

Dynamics of the spatial organization of the chromosome set in cells of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> imaginal disks normally and under the action of the tumor-inducing mutation <Emphasis Type="Italic">Merlin</Emphasis>

Fluorescence of H3-p histone and DAPI was studi ed at different stages of interphase and mitosis in cells of imaginal disks of third-instar Drosophila melanogaster larvae. Three stages differing in the spatial organization of the chromosome set in mitosis were revealed. At the first stage (prophase, prometaphase), the histone 3 phosphorylation level rises, and the volume occupied by the chromosome set in the nucleus increases. The distinctive features of the second stage (metaphase) are a gradual decrease in the histone 3 phosphorylation (the density of phosphorylation remaining constant) and a reduction of the volume occupied by the chromosome set. At the third stage (anaphase, telophase), the intensity and density of the signal from H3-p histone decrease, and the volume occupied by the chromosome set reduces. At this stage, in Mer ⁴ larvae, in contrast to the control strain, the cells prematurely pass from anaphase into telophase. In addition, a subpopulation of cells with an abnormally large volume of nuclear DNA during the G₁ period was revealed in Mer ⁴ larvae. The cells of this subpopulation do not enter into the DNA synthesis and quit the cycle.     

玉米rRNA基因的组织和表达模式

玉米（Zea mays）只有1对45S rDNA位点并在分裂期染色体形成次缢痕,是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交（fluorescence in situ hybridization,FISH）、CPD（PI与DAPI组合）染色和银染技术,分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号：荧光强烈地位于核仁周边的纽,而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出;点的数目越多,纽变得越小;点的数目多少与细胞的活性呈正相关。研究结果表明,纽代表了处于凝缩状态的非活性的rDNA染色质,纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现;不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示,大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示,同一间期细胞的2个同源rDNA位点的表达水平存在差异,同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示,早中期细胞的rDNA染色质相对解凝缩,银染在所有早中期细胞和部分中期细胞显示了明显的核仁,表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录,其转录在晚中期才停止。     

玉米rRNA基因的组织和表达模式

玉米(Zea mays)只有1对45S rDNA位点并在分裂期染色体形成次缢痕, 是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交(fluorescence in situ hybridization, FISH)、CPD(PI与DAPI组合)染色和银染技术, 分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号: 荧光强烈地位于核仁周边的纽, 而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出; 点的数目越多, 纽变得越小; 点的数目多少与细胞的活性呈正相关。研究结果表明, 纽代表了处于凝缩状态的非活性的rDNA染色质, 纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现; 不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示, 大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示, 同一间期细胞的2个同源rDNA位点的表达水平存在差异, 同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示, 早中期细胞的rDNA染色质相对解凝缩, 银染在所有早中期细胞和部分中期细胞显示了明显的核仁, 表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录, 其转录在晚中期才停止。     

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.     

Reverse fluorescent chromosome banding with chromomycin and DAPI

Two DNA binding guanine-specific antibiotics, chromomycin A₃ (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI. — In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI. — Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The CMA-banding pattern appears to be similar to the pattern found by R-banding procedures.     

Diffuse kinetochores and holokinetic anaphase chromatin movement during mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20

Mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20 was examined by light microscopy of living and fixed cells. During early prometaphase the numerous small (0.30-3.0-microns) chromosomes appear as discrete units that lack a primary constriction. However, by late prometaphase the chromosomes are tightly packed at the spindle equator and are no longer clearly resolvable as individuals. When viewed from the side the metaphase chromatin appears as a 2-3-microns wide band that spans the width of the spindle; when viewed from the pole it appears as a fenestrated disk. The metaphase chromatin splits at anaphase into two sister chromatin plates, each of which exhibits holokinetic poleward movement, i.e., all parts of each plate move as a single unit with the same velocity. In many early-to-mild anaphase cells the separating sister plates are connected by chromatin-containing bridges that break as anaphase progresses. Ultrastructural analyses of serial thick and thin sections from cells fixed by conventional, OsO4/KFeCN, or high pressure rapid freezing methods, reveal that by metaphase all of the chromosomes are interconnected to form a large, irregularly shaped fenestrated disk of chromatin. Similar analyses reveal that adjacent chromatids remain interconnected throughout anaphase. Each disk of metaphase and anaphase chromatin contains numerous kinetochores recessed within its pole-facing surface. Kinetochores consist of a fine, faintly staining fibrillar material arranged along the chromatin surface as thin (0.1-0.3 micron dia.) rods varying considerably (0.15-2.3 microns) in length. From these observations we conclude that the polycentric metaphase chromatin of A. constricta, and its holokinetic behavior during anaphase, arises from the aggregation or cohesion of smaller prometaphase chromosomes, each of which contains a single, diffuse kinetochore.     

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.     

Chromatin behaviour under influence of puromycin and 6-DMAP at different stages of mouse oocyte maturation

Preovulatory mouse oocytes were cultured in vitro up to each subsequent stages of maturation: germinal vesicle (GV), germinal vesicle breakdown (GVBD), groups of not yet individualized bivalents, circular bivalents, late prometaphase I, metaphase I, anaphase I and telophase I. The stages were identified in living oocytes by fluorescence microscopy using Hoechst 33342 as a specific vital dye. Oocytes from each stage of development developed in vitro and ovulated metaphase II oocytes were subsequently cultured in the presence of puromycin or 6-dimethylaminopurine (6-DMAP), an inhibitor of protein phosphorylation. The effects on chromatin of these drugs were studied during and at the end of culture by fluorescence and electron microscopy. We found that puromycin and 6-DMAP stop meiosis when applied at all stages of oocyte maturation, except for metaphase II. Oocytes at this stage are activated by puromycin. Reaction of the oocytes to the two drugs is different at GV and at metaphase II. All of the other stages react to the drugs by chromatin compaction, which can be followed by chromatin decondensation to form a nucleus. Our results suggest that late prophase chromatin condensation, bivalent individualization and retention of their individuality, as well as individualization of monovalents from telophase and retention of their individuality at metaphase II, are dependent on protein phosphorylation. The events occurring between metaphase I and telophase I are independent of protein synthesis and phosphorylation. The events occurring between metaphase II and formation of the nucleus are independent of protein synthesis.by U. Scheer     

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC₆ indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.     

A postprophase topoisomerase II-dependent chromatid core separation step in the formation of metaphase chromosomes

Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II. Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes. Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested. Immunolocalization of topoisomerase II alpha reveals chromatid cores analogous to those seen with silver staining. We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved.     

A comparative study of male meiosis in Drosophila melanogaster and D. virilis

Male meiosis in D. melanogaster cytologically follows the usual pattern, whereas in D. melanogaster and in D. virilis oocytes the chromosomes clump into a karyosphere at early meiotic prophase and remain so up to metaphase I.Male meiosis in D. virilis spermatocytes has an intermediate character: a part of the chromatin clumps together in a karyosphere at early prophase, whereas the other part of the chromatin remains diffuse all through prophase. At the end of prophase, the diffuse chromatin becomes integrated into the karyosphere before metaphase I. During the meiotic divisions the chromosomes have the same clumped aspect as those in Drosophila oocytes and thus differ strikingly from the dividing chromosomes in D. melanogaster spermatocytes.In D. virilis spermatocytes the nucleolus exhibits changes during the meiotic prophase that may be related to synthetical activities. The DNA specific staining with the fluorochrome DAPI reveals the existence of extrachromosomal DNA in the later prophase. Other striking differences in meiotic events between the two Drosophila species concern the centrioles and spermiogenesis.     

Telomere length measurements using digital fluorescence microscopy.

BACKGROUND: The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). METHODS: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. RESULTS: Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. CONCLUSIONS: We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.     

Distinct Dynamics of Aurora B and Survivin During Mitosis

We have studied the dynamics of Aurora B and Survivin during mitosis in living cells, using C-terminal GFP chimeras of the two proteins. These chimeras showed identical localization and behave as bona fide wild type proteins. The mobility of Aurora B-GFP and Survivin-GFP was analyzed by FRAP. The data show that Survivin-GFP, in contrast to Aurora B-GFP, is highly mobile at prometaphase and metaphase. At telophase and cell cleavage, both chimeras are found to be fully immobile. The ablation of Aurora B by siRNA results in a dramatic decrease of the Survivin-GFP mobility. These results demonstrate that Survivin, but not Aurora B, is weakly associated with the centromeric chromatin at prometaphase and metaphase. The weak association of Survivin with centromeric chromatin is dependent on the presence of Aurora B and is not affected by treatment with either nocodazole or taxol. The rapid and conditional interchange between passenger proteins that we show by live imaging indicates that the high affinity interactions demonstrated with in vitro analysis of passenger protein binding are, in fact, static “snapshots” of highly dynamic and regulated in vivo interactions in mitotic cells.     

Karyotype analysis of Nicotiana kawakamii Y. Ohashi using DAPI banding and rDNA FISH

Prometaphase cells were used to analyze the karyotype of Nicotiana kawakamii Y. Ohashi by means of sequential Giemsa/CMA/DAPI staining and multicolor fluorescence in situ hybridization with 5S and 18S rDNA. Observation of the DAPI-stained prometaphase spreads indicated that N. kawakamii had six pairs of large chromosomes, one pair of medium-sized chromosomes and five pairs of small chromosomes. The six pairs of large chromosomes possessed remarkable DAPI bands, and each could be identified from both the DAPI banding pattern and the length of the short arm. The DAPI banding pattern was approximately identical to the CMA and Giemsa banding patterns. Hybridization signals of the 18S rDNA probe were detected on two pairs of large chromosomes. In addition, two pairs of small chromosomes were identified based on the position of the 5S rDNA signals. An idiogram of N. kawakamii chromosomes was produced based on DAPI bands and rDNA loci. Received: 17 July 2000 / Accepted: 4 September 2000     

Use of DNA Fluorochromes for Studying Meiosis in the Woody Species Thryptomene Calycina

The fluorescent DNA probes DAPI and Hoechst 33258 produce superior images to the traditional acetocarmine stain of the small chromosomes of the woody shrub Thryptomene calycina at all stages of microsporocyte meiosis and microspore mitosis. Hoechst 33258 was slightly superior to DAPI because of reduced background fluorescence. Binding with the DNA-specific probes required a fixative containing chloroform to remove autofluorescent materials, a pretreatment with acetic acid and a pH of least 6 during treatment. The nucleoli did not fluoresce after treatment with DAPI or Hoechst 33258. Superior resolution of chromosomes after treatment with the fluorochromes enabled easy determination of the haploid number at metaphase I, metaphase II and at metaphase of the microspore mitosis.     

Comparison of the subunit organization of early and late replicating chromatin

A comparison was made of the subunit organization of chromatin from regions of the genome with different metaphase chromosome banding characteristics by analyzing the accessibility of early and late replicating DNA in synchronized Chinese hamster ovary cells to digestion with staphylococcal nuclease. Three measures of nuclease susceptibility were employed: (1) the release of acid-soluble material; (2) a digestion index, P, which corresponds to the proportion of internucleosome segments which experienced at least one cleavage event; and (3) the size distribution of DNA fragments isolated from digested chromatin. Little or no difference was observed in the initial rates with which nuclease converted early and late replicating chromatin to acid-soluble material, although the initial digestion rates varied with time of cell collection in the cycle (metaphase > G1 mid-S > late-S or G2). Measurements of the digestion indices of material isolated from interphase cells suggested that initial cleavage events were more rapid in early replicating chromatin than in late replicating chromatin. In contrast, electrophoretic analysis revealed that oligomer DNA fragments from early labelled metaphase chromatin were slightly larger than corresponding fragments from late labelled metaphase chromatin. The size distribution of DNA in submonomer fragments obtained from extensively digested chromatin appeared to be identical regardless of the timing of replication or cell collection. Those small differences in chromatin digestibility that were observed may reflect subtle variations in the accessibility of internucleosome regions or perhaps in the higher-order arrangement of nucleosomes. However, no gross variation in accessibility to staphylococcal nuclease digestion was observed in chromatin localized to metaphase chromosome regions with vastly different cytological staining properties.     

Use of DNA fluorochromes for studying meiosis in the woody species Thryptomene calycina.

The fluorescent DNA probes DAPI and Hoechst 33258 produce superior images to the traditional acetocarmine stain of the small chromosomes of the woody shrub Thryptomene calycina at all stages of microsporocyte meiosis and microspore mitosis. Hoechst 33258 was slightly superior to DAPI because of reduced background fluorescence. Binding with the DNA-specific probes required a fixative containing chloroform to remove autofluorescent materials, a pretreatment with acetic acid and a pH of least 6 during treatment. The nucleoli did not fluoresce after treatment with DAPI or Hoechst 33258. Superior resolution of chromosomes after treatment with the fluorochromes enabled easy determination of the haploid number at metaphase I, metaphase II and at metaphase of the microspore mitosis.     

Chromatin structure variation in successful and unsuccessful arbuscular mycorrhizas of pea

Summary Lincoln and Frisson varieties of endomycorrhiza-forming pea plants and isogenic mycorrhiza-resistant Frisson mutant (P2) plants were inoculated withGlomus mosseae. Nuclei released from inoculated and non-inoculated (control) roots were analysed for chromatin structure and activity using flow cytometric techniques. Chromatin accessibility to the specific DNA fluorochrome DAPI at saturating and non-saturating concentrations was measured. DNA fluorescence of nuclei of mycorrhizal Lincoln and wild genotype Frisson plants was significantly increased, compared to the controls, at saturating and, more strongly, at non-saturating DAPI concentrations. In contrast, the nuclei of inoculated P2 mutant roots showed a much lower increase in fluorescence, compared to uninoculated controls. Nuclei released from mycorrhiza-infected Lincoln roots were more sensitive to DNase I than those of uninfected ones. These results indicate a dramatic increase in that portion of the genome which can be transcribed in response to AM infection.Abbreviations AM arbuscular mycorrhizas - CRBCs chicken red blood cells - CV coefficient of variation - DAPI 4 6-diamidino-2-phenylindole - DNase I deoxyribonuclease I - EDTA ethylenediamine tetraacetic acid - FCM flow cytometry - TMN Tris MgCl₂ NaCl buffer     

Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI

BACKGROUND: This study investigated the relationship between chromatin compactness, which is directly related to chromatin condensation, and DAPI uptake. Materials and Methods For the structural characterization of in situ chromatin, we used fluorescence microscopy and differential scanning calorimetry on calf thymocytes. The compactness of nuclear chromatin was altered by permeabilizing native cells with NP40 detergent. A time-dependent analysis of detergent effects was performed by acquiring nuclear images at different time intervals after permeabilization. In order to compare nuclei of different sizes, we implemented a geometrical correction in the calculation of the integrated fluorescence intensity. For a quantitative evaluation of chromatin condensation we introduced two new parameters, "average chromatin packing ratio" and "average dye spatial density." RESULTS: This approach allowed us to estimate the effects of NP40 detergent at the level of in situ chromatin. Detergent effects could be modulated by changing the ionic composition of buffer. Moreover, changes of chromatin condensation induced by detergent were inversely related to modifications of nuclear volume. CONCLUSIONS: The combination of complementary information obtained by fluorescence microscopy, supported by a proper geometrical correction, and differential calorimetry allowed us to interpret the patterns of fluorescence intensities inside the nucleus in terms of chromatin structure.