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.     

Nucleolus and large nucleolar aggregates of condensed chromatin in interphase nuclei of L 929 cells

Summary Three-dimensional reconstructions show that the nucleoli from L 929 cells are associated with one or several large aggregates of chromatin displaying a honeycomb-like structure. The form and the number of both nucleoli and honeycomb structures vary as the cells emerge from the resting state and enter exponential growth. Quantitative data show that the number of honeycomb structures decreases as the number of nucleoli diminishes; both numerical regressions are significant. In addition, the nucleoli and the honeycomb structures enlarge when the cells enter the exponential growth phase.In resting cells the number of honeycomb structures is correlated to the number of nucleoli. Therefore we conclude that the large nucleolar mass of condensed chromatin, which in L 929 cells displays a honeycomb structure, contains a portion of the nucleolar organizing region.     

Nuclear proteins : IV. Deficiency of non-histone proteins in condensed chromatin of Drosophila virilis and mouse

Drosophila virilis egg nuclei were fractionated by a technique of multiple sonication and centrifugation in an isotonic buffer containing 0.15 M NaCl, Mg²⁺ and Ca²⁺. This allowed the condensed chromatin to remain tightly condensed. By Hoechst 33258 staining this procedure resulted in brightly fluorescing and poorly fluorescing fractions. The brightly fluorescing fraction was enriched in satellite DNA. Examination of the non-histone proteins by SDS slab gel electrophoresis showed that this fraction was markedly deficient in non-histone proteins and contained no unique major non-histone proteins. The poorly fluorescing fractions were enriched in non-histone proteins. Similar results were obtained with mouse liver nuclei. Comparison of the non-histone proteins of D. virilis (40% satellite DNA), D. americana (35% satellite DNA), and D. ezoana (no satellites) confirmed the absence of major, satellite specific, non-histone proteins. These results, suggesting condensed chromatin is primarily a DNA-histone complex, agree with published cytochemical studies.     

Acridine orange binding to chromatin of individual cells and nuclei under different staining conditions. I. Binding capacity of chromatin.

A study was made to find the optimal conditions for titration of the strong acridine orange binding sites of DNA in situ by an equilibrium staining method. Low concentrations of dye (≈10^?6 M) and an equilibration time of about 1 h were found necessary. Chick erythrocyte nuclei were used as a model system to compare results of this equilibrium method with those of conventional staining. Before staining, nuclei were subjected to acid extraction and denaturation or to biological activation via cell hybridization. Qualitatively similar results were obtained with the two staining methods, but the equilibrium method circumvents the problems of dye-to-dye aggregation and differences in diffusion conditions, and thus gives more easily interpretable data and true quantitative information about the properties of chromatin in situ.     

DNA constancy and chromatin structure in some cell nuclei ofAmphiuma

Synopsis In a series of microspectrophotometric and microphotometric investigations, it has been found that in lymphocytes of the primitive amphibianAmphiuma the very large amount of Feulgen-DNA per nucleus (that is, the amount of DNA revealed by a Feulgen hydrolysis-Schiff technique) is constant within the limits of the measuring error. The Feulgen hydrolysis time had to be reduced considerably in order to bring down the extinctions (optical densities) of these very voluminous and densely staining nuclei. Off-peak absorption of cells in smears stained in the usual way with the Feulgen-Schiff method appeared to be of no value in these experiments. The relation between the Feulgen-DNA content of lymphocyte nuclei of human andAmphiuma cells, as determined from the slope of the hydrolysis curve, appeared to be around 124, which fits well with biochemical data from the literature.Cytologically, a large part of the chromatin appeared to exist in large clumps of heterochromatin. In marginated plaques of condensed chromatin, local areas of lower density occur and are in close association with nuclear pores. The dense lamina is often very pronounced in these nuclei.     

Isolation, fractionation and template activity of the continuously-condensed chromatin of Euglena gracilis

A technique for isolating whole chromatin from nuclei of the lower eukaryote Euglena gracilis is presented. This chromatin, which appears under the electron microscope as uniformly condensed fibers, can, nevertheless, be subfractionated into distinct heterochromatic and euchromatic fractions. The euchromatin, comprising about 14% of the total DNA of the nucleus, contains over 80 % of the total endogenous RNA polymerase activity measured. The K_m for this enzyme is higher than that found for prokaryotes, but falls in the range found for other eukaryotes. Stability constants, calculated from cation-chromatin binding data, suggest that internal carboxyl groups of chromosomal proteins, at least, are involved in the condensation of Euglena chromatin. The relationship between Euglena chromatin and that of higher eukaryotes is discussed.     

Identification of apoptotic cells by formamide-induced dna denaturation in condensed chromatin.

In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells based on this effect. Our observations show that formamide induces DNA denaturation in apoptotic nuclei but has no such effect on DNA of non-apoptotic cells. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it possible to specifically identify the apoptotic cells. This procedure produced intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity of the formamide-MAb assay for the apoptotic cells. However, TUNEL stained 90-100% of necrotic cells in all three models of necrosis. Because the MAb did not stain cells with single- or double-stranded DNA breaks in the absence of apoptosis, we conclude that staining of the apoptotic nuclei is not influenced by DNA breaks and is induced by specific changes in condensed chromatin, such as damage to the DNA-histone interactions. Importantly, the formamide-MAb technique identified apoptotic cells in frozen sections and in histological sections of formalin-fixed, paraffin-embedded tissues.     

Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplasts completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W₁₀BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.     

Effects of Hoechst 33258 on condensation patterns of hetero- and euchromatin in mitotic and interphase nuclei of Drosophila nasuta

Embryonic and third instar larval brain cells of D. nasuta were cultured in vitro in the presence of Hoechst 33258 (H) and H + 5-bromodeoxyuridine (BUdR) for periods varying from 2 to 24 h at 24 °C. Air-dried chromosome preparations were made with and without hypotonic pretreatment and stained with Giemsa. Metaphase chromosomes from H-treated (2 h) embryonic preparations show typical inhibition of condensation of the A-T-rich heterochromatin as in mouse. Presence of BUdR with H causes inhibition of condensation in fewer embryonic metaphase cells, but in the affected metaphases the degree of inhibition is more severe. In larval brains, however, even a 24 h H or H + BUdR treatment does not cause any significant inhibition of heterochromatin condensation. It is suggested that the differences in H effect on metaphase chromosomes of embryos and larval brains is related to differences in chromosome organization in the two cell types. Exposure of H-treated embryonic as well as larval brain cells to a hypotonic salt solution prior to fixation causes a ‘supercondensation’ of the heterochromatic chromocentre in most interphase nuclei. Presence of BUdR along with H reduces the frequency of cells showing such ‘supercondensed’ chromocentre. The euchromatin region in H-treated interphase nuclei is, on the other hand, slightly more diffuse than in control nuclei. Apparently, H-binding to DNA affects the nucleoprotein organization in hetero- and euchromatic regions of interphase nuclei in specific ways.     

Control of flagellar motility in Euglena and Chlamydomonas : Microinjection of EDTA, EGTA, Mn, and Zn

When a Euglena, in a medium containing ATP, is microinjected with 7 × 10⁻¹⁴ l of 0.02 M EDTA, which binds Ca²⁺ and Mg²⁺, flagellar motility stops. Flagellar arrest in Chlamydomonas occurs with the injection of 2 × 10⁻¹⁴ l of 0.02 M EDTA. The injection of similar amounts (7 × 10⁻¹⁴ l in Euglena and 3 × 10⁻¹⁴ l in Chlamydomonas) of 0.02 M EGTA, which preferentially binds Ca²⁺, did not significantly alter flagellar motility. This suggests that a decrease in the internal Ca²⁺ concentration in Euglena or Chlamydomonas did not stimulate flagellar beating. Further, flagellar motility decreased when internal Mg²⁺ was chelated. The microinjection of Zn²⁺ into these cells caused a decrease in flagellar frequency analogous to the decrease in frequency caused by the injection of Ca²⁺ and EDTA. The microinjection of 7 × 10⁻¹⁴ l of 0.2 M Mn²⁺ caused an approx. 1.5-fold increase in Euglena flagellar motility. Chlamydomonas flagella, which cease to beat upon impalement in an Mg²⁺-free medium, resume a flagellar frequency of 18 Hz when injected with 3 × 10⁻¹⁴ l of 0.2 M Mn²⁺. In the experiments reported here, Mn²⁺ acts as an analog of Mg²⁺.     

Differential accessibility of DNA in extended and condensed chromatin to pancreatic DNase I

Pancreatic DNase I has been used to study the interaction between DNA and chromosomal proteins in extended and condensed chromatin fractions isolated from mouse and Chinese hamster livers. It was found that DNase digests extended chromatin at a faster rate than condensed chromatin, and the evidence suggests that the chromosomal proteins are more tightly complexed to the DNA in condensed than in extended chromatin. This difference in DNA-protein interaction in extended and condensed chromatin may be related to the functional difference which characterizes these fractions, and might be one of the factors underlying the production of bands on metaphase chromosomes.     

Escherichia coli plasmid pBR313 insertion into plant protoplasts and into their nuclei

Summary Cowpea mesophyll protoplasts were shown to bind irreversibly up to 3% input radioactive pBR313 plasmid DNA after 15 min of contact. Maximum uptake occurred in the presence of 5mM ZnSO₄ and 5 g/ml poly-L-ornithine. Under these conditions about one half of the TCA precipitable radioactivity was associated with the nuclear fraction and behaved as linear plasmid molecules. These could not be chased from the protoplasts upon further incubation with unlabeled plasmid DNA. The presence of donor DNA within the nuclear fraction is most probably not due to an artifactual redistribution of adsorbed plasmid DNA. Prolonged incubation periods resulted in extensive degradation of plasmid in the incubation medium but little degradation occurred in the protoplasts. The donor DNA was not covalently associated with the protoplast nuclear DNA.     

The separation of RNA polymerases I and II achieved by fractionation of plant chromatin

Chromatin-bound RNA polymerase I and II from soybean hypocotyl can be separated by differential centrifugation. While all detectable RNA polymerase I is pelleted in association with chromatin, nearly all of the RNA polymerase II activity is not pelleted even at high speeds. Substitution of pH 6 for pH 8 isolation medium results in several-fold greater recovery of chromatin-bound RNA polymerase II.     

Chromosomal localisation of DNA sequences in condensed and dispersed human chromatin.

The DNAs purified from condensed and dispersed human chromatin were used as templates for the in vitro synthesis of ³H-labelled complementary RNAs (cRNAs). These cRNAs were hybridised in situ to preparations of fixed human metaphase chromosomes which had previously been stained with quinacrine and photographed with fluorescent (UV) light. Autoradiographs of the hybridised chromosomes were stained and photographed and the results analysed by comparison of the fluorescence photographs with the autoradiographs. This method allowed positive identification of every chromosomal site of hybridisation and quantitative analysis of grain distribution over a number of metaphase spreads. The cRNA transcribed from condensed chromatin DNA (cRNA_C) hybridised mainly to a limited number of sites close to or including centromeric heterochromatin (C-bands) and also to the brightly fluorescent regions of the Y chromosome. Many of these C-band regions are known to contain satellite DNAs, indicating that the repeated DNA in the condensed chromatin fraction consists largely, if not entirely, of satellite sequences. The cRNA transcribed from dispersed chromatin DNA (cRNA_D) does not contain satellite DNAs and hybridised more generally over the chromosome arms. However, the main sites of hybridisation with cRNA_D included the C-bands in the Y chromosome and autosomes, i.e. those regions which bound cRNA_C. This suggests that nonsatellite repeated DNA sequences may be associated with satellite DNAs in the chromosomes. No general correlation between the distribution of either kind of cRNA and the overall level of quinacrine fluorescence in chromosomes or chromosome arms was detectable, nor could the dispersed fraction be equated with cytological euchromatin, since it hybridised in many sites which appear heterochromatic. However, there was a suggestion that some non-fluorescing Q-bands bound cRNA_D preferentially. The differences which were found between the distribution of the cRNAs from the two chromatin fractions may be associated with differences in genetic activity.     

Electron microscope study of chromatin in hepatocyte nuclei during the first hours after partial hepatectomy. V. Changes in the relative area of condensed chromatin and the density of chromatin fibril packing in the ultrathin sections

The degree of chromatin condensation was studied on ultrathin cell sections of guinea pig hepatocytes during the prereplicative period after partial hepatectomy. Three time points were chosen for analysis namely 2,5, 5 and 9 hrs after operation since they show marked increasing (2.5 hrs), decreasing (5 hrs) and repeated increasing (9 hrs) of the amount of ethidium bromide binding to chromatin. The degree of chromatin condensation was determined by measuring the area occupied by condensed chromatin and also by measuring the number of chromatin fibrils per a certain length. The condensed chromatin with varying localization in the nucleus were studied separately. The changes of nucleoplasmic chromatin were most pronounced: at 2.5 and 9 hrs after operation the decrease of the relative area and of the density of chromatin fibrils package was observed; these parameters were near to control at 5 hrs after operation. In general the changes in nucleoplasmic chromatin were correlated with the changes of the activity of the chromatin in the whole nucleus. The decondensation of the perimembranous chromatin was manifested in the decrease of its area and was expressed only at 9 hrs after operation. The perinucleolar chromatin was found to show the gradual decondensation which was manifested mainly by the decrease of its relative area. Thus the condensed chromatin seems to be a labile structure which undergoes essential changes in the process of the exit of the hepatocytes from G0-stage of the cell cycle, during the prereplicative period.