DNA global hypomethylation in EBV-transformed interphase nuclei.

In tumors, DNA is often globally hypomethylated compared to DNA extracted from normal tissues. This observation is usually made after extraction and exhaustive digestion of DNA followed by analysis of nucleosides by chromatography or digestion with restriction enzymes, gel analysis, and hybridization. This approach provides an average value which does not give information on the various cell subpopulations included in heterogeneous samples. Therefore an immunochemical technique was set up with the aim of demonstrating, in a population of mixed cells, the possibility of detecting the presence of individual nuclei containing hypomethylated DNA, on a cell-by-cell basis. Monoclonal antibodies to 5-methylcytidine were used to label cells grown in vitro. Under appropriate fixation and permeabilization conditions, interphase nuclei were labeled. Quantitative differences in the labeling were detected between Epstein-Barr virus-transformed cells and normal peripheral blood monocytes by flow cytometry analysis. Similar differences were observed by fluorescence microscopy. Both results were confirmed by Southern transfer and hybridization of DNA fragments generated by restriction enzyme digestion. This observation, which is in accordance with the occurrence of global DNA hypomethylation in tumors as established by chromatography, opens the field for the analysis of fresh tumor samples by flow cytometry and microscopy.     

Fine structure of interphase nuclei

The initiation and replication sites of DNA synthesis in the plasmodial nuclei of Physarum polycephalum were studied with electron microscopic autoradiography. By using both thin sectioning and whole mount techniques, it was shown that the dense chromatin masses in the nucleus consisted of predominantly elementary chromatin-like fibrils, approximately 300 Å in diameter while the electron transparent region in the nucleus consisted of predominantly finer fibrils, less than 100 Å in diameter. With electron microscopic autoradiography it was found that (1) the initiation sites of DNA synthesis were definitely in the boundary regions between the dense chromatin masses and the electron transparent region, (2) the initiation and replication sites of DNA synthesis were definitely not on the nuclear membrane, (3) within a few minutes, replication sites migrated from the initiation sites to the electron transparent region and (4) in this electron transparent region, almost all of the nuclear DNA was synthesized.     

Neutron diffraction of interphase nuclei

The neutron small-angle diffraction patterns obtained with native interphase nuclei are quite featureless and difficult to interpret. However, the difference between two patterns which have been obtained before and after having induced a change in the packing of the more stable structural entities, e.g. by a reduction of the salt concentration, shows significant changes at different scattering angles. These changes allow us to estimate the overall size of buffer-filled spaces which were formed by the separation of more stable structural entities and to establish accurate and non-destructive conditions at which changes of structure occur.Rat liver interphase nuclei have been prepared in a polyamine-containing buffer. The buffer was diluted from one-half down to one-fifth and one-tenth of its original salt concentration. In the first dilution step ($\text{1}\text{2}$ to $\text{1}\text{5}$), the difference spectrum was indicative of the formation of buffer-filled spaces with a most probable distance between a pair of scattering buffer molecules of 18 nm and with an overall size of the order of 45 to 50 nm. The second dilution step ($\text{1}\text{5}$ to $\text{1}\text{10}$) resulted in a strong increase of the intensity of the difference spectrum which had been observed in the first dilution step.     

Electron microscopy of human interphase nuclei

Quantitative electron microscopy was used to analyze surface-spread, critical-point-dried human interphase nuclei and chromatin. The following information is presented: (1) Unstimulated interphase nuclei of lymphocytes from peripheral blood have a mean dry mass of 50.30×10^?12 g. The mean dry mass of stimulated nuclei of lymphocytes was determined to be 59.34×10^?12 g, a significant statistical difference from the unstimulated ones. (2) Mean diameter of chromatin fibers and mean fiber mass per micron were 199ű15% coefficient of variation (C.V.) and 5.95×10^?16g×29% C.V., respectively. (3) A line of regression of fiber mass on fiber diameter for 83 fibers indicated that a 200-Å fiber has a mass of 5.86×10^?16g/μ, or almost the same as the mean fiber mass of 5.95× 10^?16g/μ. (4) With the value 7×10^?12g for the DNA content of an unstimulated lymphocyte nucleus, a total length of 215 cm is calculated for the DNA double helix. When this length is compared to the mean length of chromatin fiber per nucleus (7.59 cm), a ratio of 28.3 to 1 results, which is called the DNA-packing ratio. (5) This DNA-packing ratio of 28.3 is reasonably close to the packing ratio of 26.9 suggested from model calculations for the second DNA supercoil in a 200-Å chromatin fiber.     

Ultrastructural organization of freeze-fractured interphase nuclei

The morphology of intact or membrane-deprived interphase nuclei has been analysed by freeze-fracture electron microscopy. This method appears particularly useful for providing information on the distribution and organisation of chromatin and ribonucleoproteins in the absence of dehydration and embedding artifacts of conventional electron microscope techniques which, among other effects, appear to affect heterochromatin distribution, inducing its aggregation along the nuclear envelope. The main levels of chromatin superstructure, from nucleosome to solenoid fibres, are detectable in the replicas of freeze-fractured nuclei on the basis of the size of their shadow, a parameter particularly suitable for automated image analyses.     

Ultrastructural and biochemical observations on interphase nuclei isolated from chicken erythrocytes.

Adult hen erythrocyte nuclei are isolated from cells or haemolysed in situ by acting on the plasma membrane with rotating knives or with non-ionic detergents. When the isolation medium contains magnesium ions (1 mM), sucrose (0-4 M) and Tris buffer (0.01 M, pH 7-5) called SMTOG (see text), the ultrastructure in thin sections through the condensed chromatin bodies, after staining with either uranyl-lead or phosphotungstic acid (PTA), is similar to that found in the intact cell. Hence it can be concluded that the 2 phases which comprise chromatin, the o- and e-phase, survive nuclear isolation. These are so called because the structural units in chromatin are arranged at the surface of the nucleus into one or more layers and give rise to oddly (o) and evenly (e) numbered bands. The 0-phase is also largely retained after extensive washing in 0-07 M NaC1 as shown by electron microscopy and biochemical measurements; only 6% of the total nuclear protein is removed, a value small compared with the fractional amount of the chromatin protein calculated to lie in the o-phase, about 70%. After extensive washing in saline-EDTA there are structural changes in chromatin, but biochemical data show that the molecules in the o-phase are also largely retained; loss of protein amounts to between 5 and 11%. These data suggest that the o-phase is a structural component of the chromatin bodies. They support the hypothesis that condensed chromatin is formed by folding superunit threads. These units consist of a central thread-like element about 17 nm diameter which stains preferentially with uranyl-lead and forms the e-phase, with an outer cylindrical shell forming the o-phase of total diameter about 28nm. The 5-10% proteins removed by salt washes are located exclusively in a particulate component, quite likely the chromatin. They have been examined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. There are about 10 or more protein species, ranging in molecular weight from 21000 upwards. The groups of large granules previously found in the nuclear sap of intact erythrocytes are shown to be associated with an amorphous or finely fibrillar body.     

High-resolution cytometry of FISH dots in interphase cell nuclei.

BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSCM) provide indispensable tools for measuring large number of cells with low resolution. Confocal microscopy, on the other hand, is used for measuring small number of cells with high resolution. In this paper, we present a reasonable compromise between the two extremes. METHODS: We have developed a completely automated, high-resolution system (high-resolution cytometer, HRCM) capable of analyzing microscope slides with FISH-stained interphase nuclei in two dimensions as well as in three dimensions using a fully motorized epi-fluorescence microscope and a cooled digital CCD camera fully controlled by a high-performance computer which performs both acquisition and related on-line image analysis. The images of different dyes are acquired sequentially using highly specific filters and superimposed in computer memory. For each nucleus and each hybridization dot, user-selected attributes (such as position, size, intensity, etc.) are computed off-line using another processor or computer connected with a network. RESULTS: Using HRCM, it is possible to analyze multi-color preparations including UV-excited dyes as well as repeatedly hybridized preparations reacquiring individual nuclei. The speed of the acquisition and analysis is about 50 nuclei per minute in two dimensions and 1 nucleus per minute in three dimensions, but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm. CONCLUSIONS: Thus, using overnight acquisition, quantities comparable to those of FCM or LSCM measurements can be analyzed with an accuracy comparable to confocal microscopy. HRCM is suitable for a number of clinical and scientific tasks: routine diagnostics, follow-up of therapy, studies of chromatin structure, and many other different aspects of cell research.     

Automatic telomere length measurements in interphase nuclei by IQ-FISH.

To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations.     

Identification of chromosome markers in interphase nuclei

The chromosome complement of the mosquito Cuilseta longiareolata (2n=6) reveals distinguishable centromeric regions and one telomere of the Y chromosome by using light-induced differentiation and autoradiographic techniques in mitotic and premeiotic interphase nuclei. The localization of these cytological markers and their spatial relationships appear to be very similar in the two types of nuclei and suggest an interphase arrangement where centromeric regions are clustered together in a chromocenter like structure, close to the nuclear membrane, with the telomeres lying on the opposite pole of the nucleus.     

Structural organization of chromosomes in interphase nuclei

The in situ molecular hybridization method has been applied to the detection, at the electron microscope level, of SV40 viral DNA in permissively infected monkey kidney cell cultures. The observations suggest an important role of the host cell nucleolus during the lytic infection with SV40.     

Nucleolar DNA organization in the interphase nuclei of mouse fibroblasts.

The organization of nucleolar DNA in interphase nuclei of somatic cells was studied at the ultrastructural level using oxidized DAB as a nucleic acid stain. Some finely filamentous networks of DNA-containing structures were observed within the nucleolar fibrillar component. They originated from the perinucleolar shell of condensed chromatin and from a chromatinic area with a honeycomb like structure. The latter was significantly associated with nucleoli and is believed to be a part of the nucleolar organizer region.     

Localization of telomeres in plant interphase nuclei by in situ hybridization and 3D confocal microscopy

We investigated the three-dimensional (3D) arrangement of telomeres in structurally well preserved, interphase nuclei of Pisum stativum and Vicia faba root tips using in situ hybridization of a probe to telomeric sequences. The probe was labelled with either digoxygenin or biotin and hybridized sequences were detected by immunofluorescence. Three-dimensional data sets were collected by confocal optical microscopy or using a cooled CCD camera. Twelve stacks of optical sections of P. sativum nuclei and nine of V. faba nuclei were studied in detail. Projections through the stacks of optical sections revealed that, in both species, most of the telomeres were adjacent to the nuclear envelope except for a small number next to the nucleolar periphery. In V. faba nuclei, the telomeres were clearly clustered at one pole while in P. sativum there was only a slight tendency for clustering. In V. faba, clusters were found at opposite poles in pairs of sister nuclei rather than at adjacent poles as would be expected if the arrangement at telophase were maintained into interphase.by D. Bazett-Jones