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.

Study of genome organization by DNA cytochemistry and fluorescence imaging: a review.

DNA analysis by quantitative cytochemistry has been a widely exploited approach to investigate chromatin superstructural changes in relation to cell function and cell cycle progress. To this aim, a number of dyes (especially fluorochromes) for nucleic acids have been used, exhibiting different peculiarities, in terms of both binding mechanism and base specificity. Less attention has been paid to the application of different DNA staining techniques for studying possible differences in genome organization among different taxa. The purpose of this paper is to review and discuss the present reports in the literature, concerning the cytochemical analysis of genome organization, in a comparative perspective. Special attention is given to the integration of quantitative studies based on cytofluorometric and imaging techniques.

Somatovariants of Sardinian recruits from the three principal geographic areas of the island.

Using the constitutional method based on somatovariants, three samples with a total of 1207 recruits, divided according to the three main geographic areas of Sardinia (North, Center, South) were compared. Individuals from the center of the island are lighter and shorter than those belonging to the other two regions. Since this somatic typology corresponds to expectations based on data analyzed in a previous investigation, the somatotype method can be considered to be a valuable instrument for quick, preliminary constitutional typings.

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.

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.

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.

A morphotypological analysis of an Italian population: association with obesity.

The morphotypological method of Brian (1960) was applied to the data collected in Aosta (N. Italy) during an investigation on human obesity. The data consist of anthropometric measurements on members of families ascertained by the presence of obesity in the children and therefore the frequency of obesity is much higher than in the general population. One of the morphotypes was found only among the obese and two others were much more common among the obese than among the non-obese. More than 80% of the obese (vs. 10% of the non-obese) belonged to these three classes. There was a positive parent-offspring association for two of the components of the morphotype (morphy and somy).

Some parameters for a cell cycle cytotopochemical study.

With the Feulgen-Naphthol Yellow double reaction we dosed, simultaneously on a cell, [1] the DNA nuclear content (as Feulgen positive material), [2] the protein cell content and [3] the Feulgen chromatin area; the degree of condensation is expressed by the ratio [1]/[3]. The evolution of the lymphocytes culture at different times of PHA stimulation was in this way studied with the aim of defining the existence of cell subpopulations pertaining at different phases or subphases of the cycle.

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.

Human round-headed spermatozoa: a quantitative and qualitative cytochemical study on chromatin.

A quantitative and qualitative cytochemical study on human round-headed spermatozoa chromatin(Feulgen-DNA content, basic clear protein content, acid-resistance degree of DNP) was carried out. Feulgen-DNA and basic nuclear protein contents showed in different nuclear classes of various sizes within the whole population a variability of behaviour. Our data suggest the occurrence of meiotic malsegregation events followed by production of aneuploid gametes whose chromatin appears to be more compacted than controls.