Figure 1. Scheme of GC-rich isochore formation at the DNA level. During the intragenomic compositional major shifts, the ‘genome core’ from the ancestors of birds and mammals increased its GC content, allowing the formation of GC-rich isochores, which were shorter and with a much higher compositional fluctuation than the GC-poor isochores of the ‘genome desert’.

Figure 2. Chromosomal and nuclear distribution of the H3 and L1 isochores. Human chromosomes (top) and nuclei (bottom) hybridized with the GC-richest H3 and the GC-poorest L1 isochores. The H3 and the L1 isochores were labelled with biotin and digoxigenin, respectively, and detected with tetramethylrhodamine isothiocyanate (TRITC)–avidin (red signals) and fluorescein isothiocyanate (FITC)–antidigoxigenin-antibody (green signals), respectively. The yellow colour is due to the overlapping of red and green signals. Chromosomes were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). Chromosomes show the highest concentrations of H3 and the L1 isochores in telomeric and internal regions, respectively (Saccone et al., 1992; Federico et al., 2000). The nuclei show that the DNAs corresponding to the two probes are differentially located, the GC-richest regions (red signals) being more internal than the GC-poorest ones (green signals).

Figure 3. Differential compaction of chromatin in the human nuclei. In the nuclei, the GC-richest band of DNA is very decondensed compared with the GC-poorest band of DNA. This is demonstrated, for example, by in situ hybridization, in the interphase nuclei, of the DNA corresponding to the 9q34 (GC-rich) and 12q21 (GC-poor) bands (red signals), together with the relative chromosome painting (green signals). The ideograms of human chromosomes 9 and 12 at 850-band resolution show the H3⁺ (red) and the L1⁺ (blue) bands (Saccone et al., 1999; Federico et al., 2000), together with hybridization on the relative mitotic chromosome. There is a different level of compaction in the nuclei of the two compositionally opposite bands of DNAs. The upper and lower nuclei show the hybridization of the band DNA (red) and of the chromosome painting (green), respectively. In the middle nuclei, the two images overlap. The nuclei were stained with DAPI (blue). Modified from Saccone et al. (2002).

Figure 4. The gene-dense and the gene-poor regions in vertebrate cell nuclei. Interphase nuclei from Podarcis sicula and Rana esculenta were hybridized with the chicken GC-richest (red signals) and the chicken GC-poorest (green signals) DNA fractions. For a direct comparison, an interphase nucleus from a mammal (human) and a bird (chicken) species is also shown. The human cell nucleus shows the hybridization with the homologous human isochores. (a), (b), (c) and (d): Nuclei from human, chicken, lizard and frog, respectively, hybridized with the GC-rich (red signals) isochores. (a²), (b²), (c²) and (d²): The same nuclei hybridized with the GC-poor (green signals) isochores. Nuclei in the middle (a¢), (b¢) and (c¢) show the merged hybridization signals. Nuclei were stained with DAPI (blue). The bar in the bottom-left panel is 2 m. The results shown on the human and chicken nuclei are in agreement with the previously published compositional mapping of cell nuclei. Reproduced from Saccone et al. (2002).

Figure 5. Regional compositional changes in the vertebrate genome. Two chromosomes are represented in their mitotic and interphasic configurations. In warm-blooded vertebrates, the chromosomal regions with the highest concentration of genes are shown (representation of the H3⁺ bands, the genome core). In the nucleus, these regions are more open relative to the remaining gene-poor regions (genome desert). If we consider that this could be the situation also in the cold-blooded vertebrates, the GC-rich isochores could arise to enhance their thermal stability, the chromatin being highly decondensed. This stabilization was not needed in cold-blooded vertebrates, because of their lower body temperature, nor in the empty quarter of warm-blooded vertebrates, where the stability is provided by the compact chromatin structure itself. This could explain why the compositional changes were regional, instead of concerning the whole genome. Modified from Saccone et al. (2002).