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.

Non-telomeric chromosome localization of (TTAGGG)n repeats in the genus Eulemur.

The chromosomal distribution of the (TTAGGG)n telomeric repetitive sequences was studied in the Malagasy species Eulemur fulvus fulvus (2n = 60), Eulemur rubriventer (2n = 50), Eulemur coronatus (2n = 46) and Eulemur macaco (2n = 44). These sequences hybridize to the telomeres of all chromosomes of the four species and also to the pericentromeres of all chromosomes of E. fulvus, E. coronatus and E. macaco, with the exception of the pericentromeres of E. coronatus and E. macaco chromosomes 9, the homeologous E. fulvus chromosomes 2 and E. macaco chromosomes 1. In E. rubriventer only a very weak signal was detected at the pericentromeres of a few chromosomes. In E. fulvus, E. coronatus and E. macaco, non-telomeric (TTAGGG)n sequences collocalize with constitutive heterochromatin. The interspecific differences of the hybridization pattern of (TTAGGG)n sequences at the pericentromeres suggest that E. rubriventer branched off the common trunk before amplification of endogenous (TTAGGG)n sequences occurred in pericentromeric regions.

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.

Cell cycle effects of hypertonic stress on various human cells in culture.

Long-term exposure to hypertonic (HT) culture media has been found to perturb the cell cycle and change gene expression in various animal cell types. A lower growth rate, with exit of cells from the cycling compartment has been observed previously in human transformed EUE cells. The aim of this study was to investigate if the kinetic changes after long-term HT stress, were typical of transformed cells or could be also found in primary cultures of normal cells. Human transformed cells from normal and neoplastic tissues, and normal human cells of epithelial and connective origin have been studied. After the incorporation of bromodeoxyuridine (BrdUrd), the frequency of S-phase cells was estimated by dual-parameter flow cytometry of DNA content versus BrdUrd immunolabelling; the total growth fraction was also estimated, after immunolabelling with an anti-PCNA antibody. We also investigated, by polyacrylamide gel electrophoresis, changes in the amount of a 35 kDa protein band, which increased in EUE cells grown in an HT medium, and which may be directly involved in cell resistance to hypertonicity. Lower BrdUrd labelling indices and higher frequencies of cells in the G0/1 range of DNA content were common features of all the cells in HT media, irrespective of their tissue of origin; other cycle phases may also be involved, depending on the cell type considered. The mechanisms by which cells cope with the HT environment could however differ, since only some cell types showed an increase of the 35 kDa stress protein found originally in HT EUE cells.

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.

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.

A 33 kDa protein band is enhanced during long-term adaptation of EUE cells to a hypertonic medium.

A cell line derived from human embryonic epithelium (EUE cells) shows an enhanced expression of a 33 kDa protein when adapted to grow in a hypertonic medium containing 0.246 M NaCl (1.8 x the isotonic concentration). The maximum amount of this protein, followed by SDS-PAGE electrophoresis, was found after 4 days of adaptation; thereafter, the protein band remained fairly constant up to 30 days. When the cells were transferred back to a medium containing 0.137 M NaCl (isotonic medium), the protein pattern reverted to that of control cells. This protein is mainly localized in the cytosol, although a small part is associated with the 150,000 g pellet and needs detergents to be extracted. The molecular weight and the cellular location suggest a possible analogy with the so-called amphitropic proteins, that are known to interact with both the epidermal growth factor receptor and hydrophobic structures, such as the membrane phospholipids and the cytoskeletal components.

Cell kinetics of PHA-activated lymphocytes are slowed by prolonged hypertonic stress.

The effect of prolonged exposure to a hypertonic medium on human lymphocytes during mitogenic stimulation with phytohemagglutinin was investigated. The process of chromatin decondensation during the first 24 hrs stimulation (G0 to G1 transition) and the changes in kinetic parameters and the occurrence of chromosome aberrations from 48 hrs to 72 hrs of stimulation were studied. In HT medium, lymphocyte transition from G0 to G1 was slowed; there were fewer S-phase cells, after 48 hrs PHA stimulation, whereas after 72 hrs the resistant cells showed the same frequency of S-phase cells as the controls. The mitotic index was always smaller, and the frequency of G0/G1 cells larger. No significant increase in the frequencies of chromosome aberrations were found. These findings suggest that human peripheral lymphocytes can survive and grow in a hypertonic medium; chromosome damages, if not repaired, may be lethal, and only lymphocytes with normal karyotypes can survive for long times in the HT medium, although with modified kinetic characteristics.

Changes of gene expression during long term adaptation of human EUE cells to a hypertonic medium: electrophoretic protein patterns and DNase I digestion in situ.

The effects of long term (1 to 10 days) growth in a hypertonic medium have been studied in human EUE cells. Following polyacrylamide gel electrophoresis, a change in the protein pattern has been found, with the progressive enhancement, during adaptation, of a 33 kDa band. Experiments of DNA digestion in situ by DNase I showed that chromatin DNA of cells grown in a hypertonic medium is more available to the enzyme cleavage. These findings show that long term hypertonic stress is able to induce a change in gene expression in EUE cells.

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.

Effect of hypertonic medium on human cell growth: III. Changes in cell kinetics of EUE cells.

The effects of hypertonicity on cell kinetics of EUE cells in culture have been investigated. After 4 days of growth in a hypertonic medium, the plating efficiency of EUE cells was reduced and cell growth was significantly slowed. Flow cytometric measurements of DNA content in synchronized cells, as well as flow cytometric determinations of DNA content and bromodeoxyuridine incorporation in asynchronous cells, also showed that the cell cycle is slowed in a hypertonic medium. In addition, the fraction of cycling cells is smaller and their progression through the S phase slower than in an isotonic medium.

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.

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.

Sperm-chromatin maturation in the mouse. A cytochemical approach.

Cytochemical techniques were used to study chromatin during spermiogenesis and sperm maturation in the mouse, starting from the stages at which the substitution of somatic histones by testis-specific proteins occurs. It was possible to distinguish and analyze the different temporal incidence of two processes involved in sperm maturation, i.e. chromatin condensation (a tridimensional highly compacted arrangement) and chromatin stabilization (a tough structure, which protects the genome DNA). The first process, involving a reduction in the nuclear size and a decrease in the amount of sperm DNA accessible to specific cytochemical reactions and stainings, was found to reach its maximum in caput-epididymidis spermatozoa, in which electron microscopy revealed that the sheared chromatin was mainly organized into 120-A-thick knobby fibers. No further changes were found in sperm up to their appearance in the fallopian tubes. On the contrary, chromatin stabilization, the onset of which occurs in the testis (at the late spermatid stage) via the formation of -S-S- cross-links, is completed in the vas deferens, where chromatin has a superstructure consisting of thicker fibers, with diameters of 210 and 350 A. The reductive cleavage of disulfides in vas-deferens spermatozoa does not completely destroy the superstructure of sperm chromatin, which could indicate 'coiling' of the basic knobby fiber. In fact, when the ion concentration was increased, the chromatin of vas-deferens spermatozoa appeared to be organized into fibers with diameters similar to those of the caput epididymidis. This unique organization of mature sperm chromatin should have an essential role in the fast swelling of spermatozoa during fertilization.