Concetta Federico

Localization of the gene-richest and the gene-poorest isochores in the interphase nuclei of mammals and birds

At a resolution of 850 bands, human chromosomes comprise two subsets of bands, the GC-richest H3(+) and the GC-poorest L1(+) bands, accounting for about 17 and 26%, respectively, of all bands. The former are a subset of the R bands and the latter are a subset of the G bands. These bands showed the highest and the lowest gene densities, respectively, as well as a number of other distinct features. Here we report that human and chicken interphase nuclei are characterized by the following features. (1) The gene-richest/GC-richest chromosomal regions are predominantly distributed in internal locations, whereas the gene-poorest/GC-poorest DNA regions are close to the nuclear envelope. (2) The interphase chromosomes seem to be characterized by a polar arrangement, because the gene-richest/GC-richest bands and the gene-poorest/GC-poorest bands are predominantly located in the distal and proximal regions, respectively, of chromosomes, and because interphase chromosomes are extremely long. While this polar arrangement is evident in the larger chromosomes, it is not displayed by the chicken microchromosomes and by some small human chromosomes, namely by chromosomes that are almost only composed by GC-rich or by GC-poor DNA. (3) The gene-richest chromosomal regions display a much more spread-out conformation compared to the gene-poorest regions in human nuclei. This finding has interesting implications for the formation of GC-rich isochores of warm-blooded vertebrates.

Identification of the gene-richest bands in human prometaphase chromosomes.

The human genome is a mosaic of long, compositionally homogeneous DNA segments, the isochores, that can be partitioned into five families, two GC-poor families (L1 and L2), representing 63% of the genome, and three GC-rich families (H1, H2 and H3), representing 24%, 7.5% and 4–5% of the genome, respectively. Gene concentration increases with increasing GC levels, reaching a level 20-fold higher in H3 compared with L isochores. In-situ hybridization of DNA from different isochore families provides, therefore, information on the chromosomal distribution of genes. Using this approach, three subsets of reverse or Giemsa-negative bands, H3+, H3* and H3-, containing large, moderate, and no detectable amounts, respectively, of the gene-richest H3 isochores were identified at a resolution of 400 bands. H3+ bands largely coincide with the most heat-denaturation-resistant bands, the chromomycin-A3-positive, DAPI-negative bands, the bands with the highest CpG island concentrations, and the earliest replicating bands. Here, we have defined the H3+ bands at a 850-band resolution, and have thus identified the human genome regions, having an average size of 4Mb, that are endowed with the highest gene density.

The gene-richest bands of human chromosomes replicate at the onset of the S-phase.

Previous investigations on the correlations between isochore organization and human chromosomal bands have identified three sets of R(everse) bands: H3+, H3* and H3–, endowed with large, moderate, and no detectable amounts of the gene-richest H3 isochores, respectively. In the present work we compared the replication timing of these three sets of bands and showed that the chromosomal bands containing H3 isochores replicate almost entirely (in the case of H3+ bands) or largely (in the case of H3* bands) at the onset of S phase, whereas chromosomal bands not containing H3 isochores (H3– bands) replicate later. The existence, at a resolution of 400 bands per haploid genome, of at least three distinct subsets of R bands is, therefore, not only supported by their GC and gene concentration but also by their replication times.

Gene density in the Giemsa bands of human chromosomes.

The human genome is formed by isochores belonging to five families, L1, L2, H1, H2 and H3, that are characterized by increasing GC levels and gene concentrations. In-situ hybridization of DNA from different isochore families provides, therefore, information not only on the correlation between isochores and chromosomal bands, but also on the distribution of genes in chromosomes. Three subsets of R(everse) bands were identified: H3+, H3* and H3−, that contain large, moderate, and no detectable amounts, respectively, of the gene-richest H2 and H3 isochores, and replicate very early and early, respectively, in S phase of the cell cycle. Here, we investigated the GC levels, replication timings and DNA compaction of G(iemsa) bands. We showed that G bands comprise two different subsets of bands, one of which is predominantly composed of L1 isochores, replicates at the end of the S phase, has a higher DNA compaction relative to H3+ bands and corresponds to the darkest G bands of Francke (1994). In contrast, the other subset is composed of L2 and H1 isochores, has less-extreme properties in replication and composition and corresponds to the less-dark G bands of Francke.

Gene-rich and gene-poor chromosomal regions have different locations in the interphase nuclei of cold-blooded vertebrates

In situ hybridizations of single-copy GC-rich, gene-rich and GC-poor, gene-poor chicken DNA allowed us to localize the gene-rich and the gene-poor chromosomal regions in interphase nuclei of cold-blooded vertebrates. Our results showed that the gene-rich regions from amphibians (Rana esculenta) and reptiles (Podarcis sicula) occupy the more internal part of the nuclei, whereas the gene-poor regions occupy the periphery. This finding is similar to that previously reported in warm-blooded vertebrates, in spite of the lower GC levels of the gene-rich regions of cold-blooded vertebrates. This suggests that this similarity extends to chromatin structure, which is more open in the gene-rich regions of both mammals and birds and more compact in the gene-poor regions. In turn, this may explain why the compositional transition undergone by the genome at the emergence of homeothermy did not involve the entire ancestral genome but only a small part of it, and why it involved both coding and noncoding sequences. Indeed, the GC level increased only in that part of the genome that needed a thermodynamic stabilization, namely in the more open gene-rich chromatin of the nuclear interior, whereas the gene-poor chromatin of the periphery was stabilized by its own compact structure.

Genes, isochores and bands in human chromosomes 21 and 22.

The recently available DNA sequences from chromosomes 21 and 22 enabled us to define the relationships of different band types with isochores and with gene concentration and to compare these relationships with previous results. We showed that chromosomal bands appear as Giemsa or Reverse bands depending not on their absolute GC level, but on the composition GC level relative to those of adjacent contiguous bands. We also demonstrated that the GC-richest, and gene-richest H3+ bands are characterized by a lower DNA compaction compared with the GC-poorest, gene-poorest L1+ bands. Moreover, our results indicate that the human genome contains about 30,000 genes.

The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

In the nuclei of human lymphocytes, chromosome territories are distributed according to the average gene density of each chromosome. However, chromosomes are very heterogeneous in size and base composition, and can contain both very gene-dense and very gene-poor regions. Thus, a precise analysis of chromosome organisation in the nuclei should consider also the distribution of DNA belonging to the chromosomal bands in each chromosome. To improve our understanding of the chromatin organisation, we localised chromosome 7 DNA regions, endowed with different gene densities, in the nuclei of human lymphocytes. Our results showed that this chromosome in cell nuclei is arranged radially with the gene-dense/GC-richest regions exposed towards the nuclear interior and the gene-poorest/GC-poorest ones located at the nuclear periphery. Moreover, we found that chromatin fibres from the 7p22.3 and the 7q22.1 bands are not confined to the territory of the bulk of this chromosome, protruding towards the inner part of the nucleus. Overall, our work demonstrates the radial arrangement of the territory of chromosome 7 in the lymphocyte nucleus and confirms that human genes occupy specific radial positions, presumably to enhance intra- and inter-chromosomal interaction among loci displaying a similar expression pattern, and/or similar replication timing.

Phenylurea herbicides induce cytogenetic effects in Chinese hamster cell lines

The intensive use of herbicides over the last few decades has caused a general increase of environmental pollution. It is thus very important to evaluate the possible genotoxic properties of these chemical compounds as well as identifying their mode of action. Phenylurea herbicides are selective agents widely used for the control of infestant plants. Of these herbicides, which are widely used in agriculture, we analysed four of the less intensively studied molecules. More precisely, we investigated the genotoxic effects of fenuron, chlorotoluron, diuron, and difenoxuron by analyses of chromosomal aberrations (CAs) and sister chromatid exchange (SCE) in exposed mammalian cells. We used the Chinese hamster ovary (CHO) and epithelial liver (CHEL) cell lines, endowed with the absence or the presence, respectively, of an enzymatic system to activate pro-mutagenic compounds. Our results show that all herbicides tested induce, at high concentrations, an increasing number of CAs in non-metabolising CHO cells. Instead, in the exposed CHEL cell line, the four herbicides induced CAs also at the lowest dose-level. In the CHEL cells, a statistically significant increase of SCE was also observed. The phenylurea herbicides showed direct genotoxic activity, but the cytogenetic effects were greatly enhanced after metabolic conversion. These data, together with other information on phenylurea herbicides, are of great interest from the environmental point of view, and for human health. In fact, intensive use of herbicides contaminates soil, surface water, groundwater and agricultural products, and thus should be taken in particular consideration not only for those initiatives to specifically protect exposed workers, but also to safeguard the health of consumers of agricultural products.

Different Retinal Expression Patterns of IL-1α, IL-1β, and Their Receptors in a Rat Model of Type 1 STZ-Induced Diabetes.

Diabetic retinopathy (DR), a common complication of diabetes, remains a major cause of blindness among population. Considerable amounts of evidences suggest the involvement of inflammatory process in this pathology. Increased levels of proinflammatory cytokines, including interleukin-1β (IL-1β), were found in the vitreous of diabetic patients and in the retina of diabetic rats. However, in this context, no attention has been given to the other main IL-1 family members: IL-1α, two transmembrane receptors IL-1RI and IL-1RII and the natural antagonist receptor IL-1Ra. Despite that they actively participate in the IL-1-mediated inflammation process, their implication in DR has not been described. Thus, we investigated by Western blot and confocal laser scanning microscopy analysis the effect of hyperglycemia on expression of IL-1 family members in retinal layers, using an in vivo model of type 1 diabetes. It was induced in adult rats by intraperitoneal injection of streptozotocin (STZ). Exposure to hyperglycemia induces a significant increase in the protein expression of IL-1β, IL-1RI, IL-RII and IL-1Ra but not of IL-1α. Moreover, high glucose alters their distribution pattern in the rat’s retinal layers. Among these latter, the most compromised are the photoreceptor, the inner plexiform and ganglion cell layers. These findings support previous data demonstrating the involvement of inflammation in DR and suggest new pharmacological approaches for the treatment of this pathology.

Avian genomes: different karyotypes but a similar distribution of the GC-richest chromosome regions at interphase

The chicken karyotype, like that of the vast majority of avian species, shows a large number of dot-shaped microchromosomes that are characterized, like most telomeric regions of the macrochromosomes, by the highest GC levels and the highest gene densities. In interphase nuclei, these gene-dense regions are centrally located, and are characterized by an open chromatin structure (a similar situation also exists in mammals). Avian species belonging to the Accipitridae family (diurnal raptors) show a karyotype with no very large chromosomes, and with only a very small number of microchromosomes. To identify the GC-rich (and gene-rich) regions of the chromosomes and nuclei from Accipitridae, we performed heterologous in-situ hybridizations using chicken GC-richest isochores as probes. Our results clearly show that the gene-rich regions are prevalently located in the few microchromosome pairs and in the telomeric regions of the middle-sized chromosomes, as well as in the interior of the interphase nuclei. This result is consistent with a common organization of the genome in the nuclei of warm-blooded vertebrates. Indeed, in spite of the different size and morphology of the chromosomes, the gene-dense regions are always located in the interior of the nuclei.