Expression of cell cycle related proteins--proliferating cell nuclear antigen (PCNA) and statin--during adaptation and de-adaptation of EUE cells to a hypertonic medium.

EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle-related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/1 range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the G0/1 range of DNA contents: this suggests that a G0 block occurs under long-term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.

Proliferating cell nuclear antigen (PCNA)/cyclin expression during the cell cycle in normal and leukemic cells.

Bivariate flow cytometric analysis of the cell proliferation-associated nuclear protein, identified as the "proliferating cell nuclear antigen" (PCNA)/cyclin and of nuclear DNA content, was performed in quiescent and mitogen-stimulated human peripheral blood lymphocytes, in EUE (human embryonic epithelium) cells, before and after a long-term exposure to a hypertonic (HT) medium, in 4 human leukemic cell lines and in fresh bone marrow (BM) cells from 10 patients with untreated acute non-lymphoblastic leukemia (ANLL). The PCNA/cyclin was detected using both an autoantibody extracted from sera of systemic lupus erythematosus patients and the recently produced mouse monoclonal antibody (MoAb) IgG, named 19F4. The distribution of cells in the different phases of the cycle and the percentage of S-phase cells were obtained in duplicate samples, by DNA flow cytometry (FCM) and by dual parameter FCM of DNA content and bromodeoxyuridine (BUDR) incorporation. In all cell types, the non-specific cytoplasmic background fluorescence was significantly lower with the MoAb compared to that obtained with the polyclonal Ab. The percentage of PCNA-positive cells (both with the autoantibody and the 19F4 MoAb) was always higher than that of S-phase cells by DNA FCM and of BUDR-labeled cells. The pattern of PCNA-expression in both normal proliferating cells and acute leukemia cells, showed that most G0/G1 cells did not express significant amounts of PCNA; an increase in PCNA immunofluorescence was found in late G1 cells, and further increases were observed in S- and G2-M phase cells. PCNA/cyclin, as revealed both with autoantibodies and with the 19F4 MoAb, is associated with all actively or potentially dividing (i.e. G1, S and G2-M) cells thus identifying the proliferative cellular compartment. Combined with the use of multiparameter FCM techniques, the PCNA immunolocalization offers a useful tool to study cell kinetics in normal and leukemic human cell populations.

Facts and paradoxes in current notions of nuclear organization and function.

Invisible compartments, identified rather by their activities than by their morphology, seem to operate in the nucleus. These compartments interrelate somehow, including mediation by the nuclear matrix. As our knowledge about the nucleus increases, more paradoxes become evident. We here consider some of them: 1) the well-known C-paradox of Cavalier-Smith, concerning the disproportionate amount of nuclear DNA content in comparison with the amount of DNA potentially able to transcribe; 2) the DNA folding in the chromatin fibre and its superorganization within the nucleus, which seems to be in opposition with the transcribing and self-replicating activities; 3) the elusive role of the DNA sequences with different degrees of repetitivity; and 4) the compartmentalization in the nucleus and how it relates to transcription, processing and transport of transcripts, and to DNA reduplication. We conclude by introducing the concept of species specific, minimal, but essential genome components, i.e. the elusive few thousand DNA bases that, in our hypothesis, act as a functional bridge between the nuclear matrix and chromatin.

Immunoelectron microscopical distribution of histones H2B and H3 and protamines in the course of mouse spermiogenesis.

We have followed the fine structural distribution of two nucleosomal core histones, H2B and H3, and of protamines in the course of mouse spermiogenesis by means of specific antibodies and ultrastructural immunocytochemistry. Our results demonstrate that the nuclear labeling density of histone H2B decreases during steps 6-8 and then increases again in step 9-10 spermatids, while the labeling for histone H3 is constant throughout this period. In step 12 spermatids, the anti-H2B antibody labels mainly the central area of the nucleus. The first signs of protamine labeling are present in step 12 spermatids, where the gold grains can be found over the periphery of the nucleus. Later on, protamine labeling constantly increases and, by the end of spermiogenesis, the whole nucleus is labeled. We suggest that the morphological and structural differences between the central area and the periphery of mouse spermatids are, at least partly, due to a difference in the protein moiety associated with DNA. The central area, which is peculiar to the mouse and has been previously considered as a focus of chromatin condensation, represents, however, the last nuclear region containing histones and consequently the last area where the substitution of histones by protamines takes place.

Occurrence of DNA sequence differences in C-heterochromatin of Eulemur coronatus and Eulemur macaco, as revealed by fluorescence resonance energy transfer.

In the genus Eulemur (Malagasy lemurs) karyotype diversification has occurred mainly through Robertsonian mechanisms of chromosome fusion (Rumpler et al., 1976). Eulemur coronatus is the sole species to have the largest genome size, due to a very large amount of C-heterochromatin, mostly located at the pericentromeric regions of the largest chromosomes (Warter and Rumpler, 1985). This increase in C-heterochromatin was thought to be due to DNA amplification (Ronchetti et al., 1993). The aim of this work was to investigate whether the large C-heterochromatin of Eulemur coronatus might have derived by amplification of the smaller C-heterochromatin of Eulemur macaco, a closely related species with smaller genome size. To obtain information on the overall base composition of the total genomes, on the relative interspersion of AT and GC base paris along the DNA molecule and on the structural differences in C-heterochromatin, we used a quantitative cyto-chemical approach, based on fluorescence resonance energy transfer (FRET) between DNA-specific fluorochromes (i.e. the AT-specific Hoechst 33258, and the non base-specific dye, propidium iodide). Micro-spectrofluorometry and image analysis were used to investigate both the overall FRET efficiency and its spatial distribution along the chromosome arms. FRET efficiency values of the DNA in C-heterochromatin were significantly different in the two Eulemur species, indicating a different qualitative composition of repetitive DNA. This suggests that the repetitive DNA of Eulemur coronatus cannot have originated by amplification in toto of the repetitive DNA sequences of Eulemur macaco.

[Some cytochemical characteristics of the chromatin in the erythrocytes of Xenopus laevis Daud]. / Su alcune caratteristiche citochimiche della cromatina negli eritrociti de Xenopus laevis Daud

The Xenopus laevis Daud. genome offers a stimulating model of a chromatin (Davidson et al., 1975) in which a high ratio of repetitious DNA sequences has been recorded (45% of total genome according to Davidson et al., 1975). We have studied cytochemically this model "in situ", on the erythrocytes of air-dried smears of peripheral blood. The cells, which are non-dividing and not engaged in DNA reduplication, constitute almost hypothetically a homogeneous class constantly in G1 phase of the cellular cycle. Our methods are: a) analysis on the curves of Feulgen hydrolysis kinetics to attempt to find the depurination and depolymerisation patterns of DNA according to Andersson et al. (1975); b) determination of the DNA resistence ratio to chemical denaturation with a quantitative relative microdensitometric determination of methyl-green staining, according to Scott (1967) specific technical conditions. For comparison, qualitative and preliminarly subjective control determinations have been made with acridine-orange in correspondent denaturation conditions. Both the a) and b) methods have been performed with and without previous HCl dehystonisation and with and without formaldehyde pretreatment. The results with the a) and b) methods have not yet been theoretically compared and the possibility of an approach to this correlation is discussed here as a tool for a possible cytochemical quantitative measure of the nuclear fraction of repetitious DNA "in situ".

A histochemical approach to the knowledge about the neuron nucleus: the "pre-alarm chromatin".

Chromatin as a functional whole. Since the nineteen-fifties (1,2), studies on the histochemistry of the nucleus have been based on its concept as a whole: measurement of the DNA content, and the ratio between nucleus size and cell size appeared to be (and were in effect) an indication of the functional status of the single cell and of the cell population. Two decades later, the already well-known morphological distinction between the chromatins as euchromatin and heterochromatin was reinterpreted on the basis of the degree of spiralization of the nucleosomal fiber and its complexity (3). Subsequently, considerable information about the non-random interphasic position of the chromosomal domains in the nucleus was obtained by in situ hybridization, and the successive reconstruction of their location in the nucleus by image processing with Normarski optics and rotating stage or by confocal microscopy (4-8). Moreover, immunological studies using monoclonal antibodies raised against the splicing factors acting on nuclear pre-mRNAs in discrete nuclear regions (spliceosomes) (9,10), lent support to the notion that the chromatin machinery operates as a whole.

Genome size and qualitative and quantitative characteristics of C-heterochromatic DNA in Eulemur species and in a viable hybrid.

The amounts of nuclear DNA and the AT and GC content of four Eulemur (Prosimii, Lemuridae) species and of an E. coronatus x E. macaco hybrid were measured by flow cytometry in peripheral blood leukocytes, following propidium iodide, Hoechst 33258, and mithramycin staining. Hoechst 33258 and mithramycin were also used to evaluate the base composition of genomic DNA in the chromosomes. The amount of DNA resisting C-banding pretreatment (C-heterochromatic DNA) was measured in metaphase chromosomes by static fluorometry. The genome of E. coronatus was significantly larger than the genomes of all other species examined, due to a higher content of pericentromeric, mainly GC-rich, heterochromatic DNA. The restriction banding patterns produced by BamHI digestion and ethidium bromide staining on extracted DNA were studied in the hybrid and its parental species (E. coronatus and E. macaco). The restriction banding pattern of the sole E. coronatus individual showed two bands which were repeated in the restriction banding pattern of the hybrid. The qualitative and quantitative differences of C-heterochromatic DNA in E. coronatus confirm the "splitting" processes and the phylogenetic relationships in the genus Eulemur suggested by Jung et al. (1992) on the basis of the restriction banding patterns produced by endonuclease digestion.

Genome size and constitutive heterochromatin in Hylobates muelleri and Symphalangus syndactylus and in their viable hybrid.

Genome size was measured as the amount of Feulgen-stained DNA in six species of the family Hylobatidae and in a hybrid of the gibbon (Hylobates muelleri) and siamang (Symphalangus syndactylus). The family, on the whole, exhibits a wider range of genome sizes than pongids; in particular, the siamang has about 15% more DNA than the 44-chromosome Hylobates species of the "lar" group. Quantitative analysis of C-heterochromatin in hybrid metaphases showed that the difference in genome size of the parental species correlates with the amount of C-band-positive material. Hylobatids are the only group of primates in which karyotype diversification has taken place with a massive quantitative change in constitutive heterochromatin.

Cytochemical evaluation of C-heterochromatic-DNA in metaphase chromosomes.

A method is proposed to evaluate the amount of DNA resistant to the C-banding pretreatments (C-heterochromatic-DNA) in metaphase chromosomes. Measurements were performed by microfluorometry on propidium iodide stained metaphases of man, gorilla and mouse; in these species, C-heterochromatin exhibits significant differences of both base composition and distribution along the chromosomes. The amount of C-heterochromatic-DNA was found to be about 16%, 28% and 58% of the total DNA content (genome size) in man, gorilla and mouse, respectively. The areas of C-bands after Giemsa staining were also assessed by microdensitometry, and corresponded to about 8%, 15% and 14% of the total karyotype area of man, gorilla and mouse respectively. No direct relation thus exists between C-band areas and the amount of DNA resistant to the C-banding pretreatments. In man and gorilla, the amount of C-heterochromatic-DNA accounts for the differences observed in genome size.

The effect of different fixatives on chromatin: cytochemical and ultrastructural approaches.

This study explores the effects of two types of fixative on chromatin. The first type (acrolein, glutaraldehyde) engenders a high degree of ultrastructural preservation. The other type are fixatives that are widely used in cytochemistry and cytogenetics (acetic acid, 3:1 by vol. methanol-acetic acid, methanol alone, formaldehyde). Lymphocytes of adult rats so-fixed in vitro were prepared for electron microscopy or microdensitometric evaluations of smears. Assessments were made of variations in their total protein, nuclear basic protein and DNA contents. DNA was determined both as Feulgen-positive material and by its binding of intercalating dyes (Methyl Green, specific for double-stranded polynucleotides). Our results showed that some fixatives break up the chromatin organization by acting on particular components of chromatin fibres. They can thus be considered to be destructive agents in situ. In addition, a revaluation of some aldehyde fixatives is proposed for both ultrastructural and cytochemical research.

Analysis of some cytotopochemical parameters for a definition of the chromatin status.

A study of the curves of Feulgen hydrolysis kinetics has been performed on different cell types of the same animal species and on the same cell type from different species at different taxonomical level. The curves were analyzed by a computer programme of least square fit to the Bateman's function, which gives a description of DNA hydrolysis kinetics by three parameters only: 1) amount potentially stainable groups; 2) depurination rate constant; 3) depolymerization rate constant. The chromatin homogeneity within the nucleus seems to affect these parameters, together with the degree of chromatin compaction. Furthermore the thermal denaturation (100 degrees C), also after fractionate histone extraction, shows a resistant DNA fraction, which does not appear to be affected by the degree of chromatin compaction only.

Kinetics of DNase I digestion of interphase chromatin in differentiated cell nuclei of the mouse: a flow cytometric study.

The process of DNA digestion with DNase I was monitored in interphase chromatin of differentiated cells by flow cytometry after DNA staining with either the intercalating dye propidium iodide (PI) or the AT specific dye Hoechst 33258 (HO). Nuclei from the liver, kidney and spleen of the mouse were studied after different digestion times (0 to 120 min). During the first 30 min of treatment, a tissue specific digestion pattern was found after PI staining; from 60 min onward, the digestion curves ran parallel, with minor quantitative differences among the cell types. After HO staining, the digestion kinetics appeared to be similar for all the cell types; this is likely due to the peculiar base composition of the mouse genome, where inactive c-heterochromatin is exceptionally AT-rich. No quantitative correlation was found between interphase "heterochromatin" and chromatin DNA which is resistant to DNase I cleavage, while the amount of DNase-I-sensitive DNA does not correspond to the interphase "euchromatic" component. It was confirmed that the flow cytometric approach is a tool for quantifying relative changes in the functional state of chromatin in differentiated cell systems.

Cytofluorometric study of nuclear sulphydryl and disulphide groups during sperm maturation in the mouse.

The amount of nuclear sulphydryl and disulphide groups was determined by cytofluorometry on single mouse spermatozoa from the caput, corpus and cauda epididymidis, and the vas deferens. N-(7-Dimethylamino-4-methylcoumarinyl) maleimide was used as a specific and quantitative fluorescent reagent for thiols. The sperm nuclear content of free sulphydryl groups decreased sharply from the caput epididymidis to the vas deferens. The amounts of cysteine residues present as the thiol form in the spermatozoa from the caput, corpus and cauda epididymidis and vas deferens were 50, 15, 5 and 3% respectively.