C. Sentís

Morphology of the axial structures in the neo-XY sex bivalent of Pycnogaster cucullata (Orthoptera) by silver impregnation

A simple method, using silver impregnation after 2 x SSC pretreatment allowed us to demonstrate axial structures (cores) in the metaphase I bivalents of the neo-XY race of Pycnogaster cucullata under bright-field microscopy. Axial structures can also be shown in DNA-depleted metaphase I bivalents, suggesting that DNA is not essential to demonstrate these elements. Specifically differentiated regions, which coincide with kinetochores, secondary constrictions (including the NOR), and chiasmata were also found. These regions have a characteristic morphology and therefore may be utilized for cytogenetic analysis. The simultaneous visualization of all these regions allowed us to establish their spatial relationships and hence the basic structural organization of the neo-XY sex bivalent in this species.

Visualization of R-bands in human metaphase chromosomes by the restriction endonuclease MseI

Human metaphase chromosomes were treated with the restriction endonuclease MseI, which cuts DNA at TTAA sequences. This enzyme preferentially cuts and extracts DNA from G-bands and thus is the first restriction endonuclease allowing direct R-band visualization. Specific patterns ranging from R+C-like to C-like banding can be induced, depending on the concentration of the enzyme. At intermediate concentrations, only a subset of R-bands are produced, corresponding to GC-rich bands that are especially resistant to heat denaturation (so-called T-bands). These results suggest that compositional differences between chromosomal regions determine the different rates of cleavage by MseI, not only between R- and G-bands but also among different R-bands.

C-heterochromatin polymorphism in Baetica ustulata: intraindividual variation and fluorescence banding patterns

In an attempt to determine the nature and extent of the extensive C-banding heterochromatin variation found in the monospecific genus Baetica, 52 individuals were analysed by C-banding and fluorescence methods. The use of fluorescent DNA-binding dyes with different specificities allowed us to distinguish three major categories of constitutive C-heterochromatin. Two of these correspond to AT-rich and GC-rich DNA; the third shows no fluorescence with any of the fluorochromes employed. Apart from confirming polymorphisms for C-band patterns in the medium-sized autosomes, several C-banding variants (cytotypes) were found in the two testes of one individual. From a comparative study of these cytotypes we deduce that equal or unequal sister chromatid exchanges, as well as mitotic crossovers, operating during the early germ line divisions have been the major mechanisms responsible for this intraindividual variation. Because of the high level of C-band polymorphism found in this species, it seems reasonable to argue that, for unknown reasons, certain processes that normally occur over generations have occurred repeatedly in this abnormal individual.

Non-uniform distribution of methylatable CCGG sequences on human chromosomes as shown by in situ methylation

We carried out in situ methylation of human chromosomes with the HpaII methylase using [3H]methyl-S-adenosyl-l-methionine as the methyl group carrier. Autoradiographs localising [3H]methyl groups show methylatable CCGG sequences in the R-bands as well as in the short arms of the acrocentric chromosomes that include ribosomal DNA. The strongest labelling was observed over a subset of R-bands, including T-bands. Since methylatable CCGG sequences are representative of the unmethylated fraction of DNA, we suggest that differences in the degree of DNA methylation could be involved in the structure and function of chromosomal bands.

In situ digestion of satellite DNA of Scilla siberica

Restriction endonucleases have been used to digest DNA in fixed metaphase chromosomes of animal species. However, constitutive C-heterochromatin of plant species is resistant to these enzymes suggesting that the special structural organization of plant C-bands is an impediment to the activity of restriction endonucleases. In order to test this hypothesis, we have chosen the species Scilla siberica, whose purified satellite DNA, localised at the heterochromatic regions, is extensively digested by HaeIII. In situ treatment with HaeIII alone does not produce significant digestion of heterochromatin, but subsequent treatment with proteinase K results in extensive digestion of heterochromatic regions producing unstained gaps. These results indicate that HaeIII is able to access and cut chromosomal DNA from C-bands, but the DNA fragments remain attached to chromosomal proteins that characterize the complex structure of heterochromatin in this species. Although there are no reasons to suppose that accessibility of chromosomal DNA of S. siberica to restriction enzymes can be impeded, it would be reasonable to think from our results that some special features of heterochromatin organization in plants contribute to the formation of a complex structure that makes chromosomal DNA extraction impossible.

Longitudinal differentiation of the human Yq heterochromatin as revealed by the restriction enzyme TaqI.

The restriction endonuclease TaqI cleaves DNA at TCGA sites which are very common in human satellite DNAs. However, this enzyme was not used successfully up to now to digest constitutive heterochromatin of human chromosomes, where those highly repetitive DNAs are preferentially located. In this work, we show that TaqI is able to cut and extract DNA from the major heterochromatic regions on chromosomes 1, 9, 15, and 16 which appear as unstained gaps. Yq heterochromatin displays moderate digestion along its entire length but a middle region can be distinguished which is usually more affected. Complete digestion of Yq heterochromatin can be achieved when this block has been previously undercondensed by treating cell cultures with the cytidine analog, 5-azacytidine. Thus, it may be deduced that some factors related to chromatin organization might be involved in the action of TaqI. These results come to reinforce previous data about heterogeneity of Yq heterochromatin, and allow us to subdivide it into three different regions according to their differential response to TaqI digestion.

In situ methylation of insect chromosomes with methylase HpaII

A method of in situ DNA methylation with the prokaryotic methylase HpaII has been developed on fixed mitotic and meiotic chromosomes of the insect species Baetica ustulata. Incorporation of methyl groups into the chromosomal DNA is revealed by autoradiography using a labelled substrate and by its ability to prevent endonuclease digestion. The method allows direct visualization of clusters of methylatable CCGG sites. The distribution of these clusters in the chromosome complement of Baetica shows two separate domains of heterochromatic DNA which differ in their methylation patterns. Each is distributed at equivalent locations in both homologous and nonhomologous chromosomes. The existence of two compartments, one methylated and the other unmethylated, in the heterochromatic DNA could be interpreted as a remnant of the ancestral echinoderm-like pattern of methylation.

Distribution of DYZ2 repetitive sequences on the human Y chromosome

The distribution of the 2000 copies of the Y-specific repetitive family DYZ2 is controversial since previous reports have mapped these sequences to different sites of the Yqh region. In this work, we have performed non-radioactive in situ hybridization of a cloned DYZ2 fragment at higher stringency conditions on 5-aza-cytidine-enlarged Y chromosomes; the results suggest a non-uniform distribution of these sequences, which are preferentially located at the proximal and distal parts of Yqh, including the C+/Q-heterochromatin at the boundary with the euchromatic region.

Latent NORs in the species Pycnogaster cucullata (Orthoptera)

The chromosomal location of the ribosomal cistrons 18 + 26S and 5S has been determined in spermatocytes of two cytological races of Pycnogaster cucullata by in situ hybridization using molecular probes from both types of rDNA. A comparative analysis with previous results suggested the existence of more 18 + 26S rDNA sites than active NORs as shown by silver impregnation, but 5S rDNA sites are outside the NORs. Thus, different categories of latent NORs have been shown in this specialised cell type which are discussed in relation to the evolutionary process of differentiation in this species.

Differential sensitivity of constitutive and facultative heterochromatin in orthopteran chromosomes to digestion by DNaseI

In situ pancreatic DNaseI digestions were used as probes to study the structural organization of facultative and constitutive heterochromatin during both mitotic and meiotic divisions. Three different types of heterochromatic regions from three insect species were chosen for this study. These regions had been previously characterized by in situ treatments with restriction endonucleases (AT and GC rich DNA sequences). Progressive increase in DNaseI concentration (from 10 to 200 ng/ml) or in incubation time (from 5 to 30 min) revealed a specific pattern of sequential digestion of the constitutive heterochromatic regions, the centromeric ones (AT-rich DNA) being the most resistant to DNaseI action. The interstitial C-bands (with AT or GC-rich DNA) were more sensitive to DNaseI, and the band 4.4 from Baetica ustalata was the most resistant of the non-centromeric bands. Similar results were obtained during meiosis, but increased accessibility to DNAseI was observed compared to mitosis. DNA methylation in the non-centromeric band 4.4 of B. ustulata could be responsible for its differential digestion with respect to the remaining intercalar heterochromatin. Facultatively heterochromatic regions (X chromosomes) were found to exhibit a differential response to DNaseI attack from mitosis to meiosis. While they behaved as cuchromatin during mitosis, they were the most resistant together with centromeric heterochromatin regions, during metaphase I and II. The different responses to digestion of the X chromosome and X-derived regions between somatic and meiotic divisions are probably a consequence of the changes in the organization of this chromosome during the facultative heterochromatinization process.