F. Pelliccia

Ag staining of the nucleolus organizer (NO) and its relationship to satellite association

SummaryThe frequency of involvement in satellite association and the frequency of selective staining of the secondary constrictions with silver solutions have been studied in five phenotypically normal individuals, all carriers of morphological variants of the nucleolus organizing region (NOR). The results show the preferential involvement of some morphological markers in satellite association, and also their preferential staining with Ag-I. It has also been shown that acrocentric chromosomes involved in satellite association are always stained by silver.

Characterization of the human common fragile site FRA2G.

Common fragile sites are nonrandom loci that show gaps and breaks when cells are exposed to specific compounds. They are preferentially involved in recombination, chromosomal rearrangements, and foreign DNA integration. These sites have been suggested to play a role in chromosome instability observed in cancer. In this work we used a FISH-based approach to identify a BAC contig that spans the FRA2G fragile site located at the 2q31 region. Our observations indicate that a very fragile region spanning at least 450 kb is present within a large fragile region that extends over 1 Mb. At least seven genes are mapped in the fragile region. One of these seems to be a good candidate as a potential tumor suppressor gene impaired by the recurrent deletions observed at the 2q31 region in some neoplasms. In the fragile region, a considerable number of regions of high flexibility that may be related to the fragility are present.

Chromosome length and DNA loop size during early embryonic development of Xenopus laevis

The looped organization of the eukaryotic genome mediated by a skeletal framework of non-histone proteins is conserved throughout the cell cycle. The radial loop/scaffold model envisages that the higher order architecture of metaphase chromosomes relies on an axial structure around which looped DNA domains are radially arranged through stable attachment sites. In this light we investigated the relationship between the looped organization and overall morphology of chromosomes. In developing Xenopus laevis embryos at gastrulation, the bulk of the loops associated with histone-depleted nuclei exhibit a significant size increase, as visualized by fluorescence microscopy of the fully extended DNA halo surrounding high salt treated, ethidium bromide stained nuclei. This implies a reduction in the number of looped domains anchored to the supporting nucleoskeletal structure. The cytological analysis of metaphase plates from acetic acid fixed whole embryos, carried out in the absence of drugs inducing chromosome condensation, reveals a progressive thickening and shortening of metaphase chromosomes during development. We interpret these findings as a strong indication that the size and number of DNA loops influence the thickness and length of the chromosomes, respectively. The quantitative analysis of chromosome length distributions at different developmental stages suggests that the shortening is timed differently in different embryonic cells.

Silver staining of the nucleolus organizer regions (NOR) requires clusters of sulfhydryl groups.

Silver stainability of nucleolus organizer regions (NORs) appears to be correlated with the presence of grouped sulfhydryl (SH) side chains of proteins. In fact, heavy metals with high affinity for SH groups, such as Hg and Cu, do prevent the silver staining reaction. Ferricyanide, which is known to oxidize SH to disulfides, also prevents any further reaction with silver. On the other hand, alkali and reducing agents (mercaptoethanol, cyanide) do not affect silver stainability of the NORs. These results show that the silver staining reaction is not related to disulfide or persulfide groups and that alkali-soluble, acidic nuclear proteins per se do not play a major role in this process.

Molecular characterization of the human common fragile site FRA1H.

The molecular basis of the fragility of common fragile sites (CFS) and their role in chromosome instability and in altered expression of associated genes in cancer cells have not yet been clarified. In the present work we analyzed the human CFS FRA1H. FRA1H is the first characterized CFS the expression of which is not induced by aphidicolin but instead by DAPI. 5‐azaC, 5‐azadC, and Ad12 induce a CFS with the same cytogenetic location. By using FISH analysis with BAC clones, we determined that this CFS extends for approximately 10 Mb, and is therefore one of the largest characterized CFSs. FRA1H maps to the chromosome bands 1q41 and 1q42.1 thus spanning an R‐band/G‐band boundary, a region considered difficult to duplicate. The FRA1H DNA sequence was analyzed to identify coding sequences, the AT content, the type and quantity of the DNA repeats, the CpG islands, the matrix attachment regions, and the number and distribution of high‐flexibility regions. A 120 kb long sequence was identified that is very AT‐rich (64.6%), has a very large number of flexibility peaks and that may be involved in inducing fragility in the surrounding regions. Among the other genes, two very large genes (USH2A, ESRRG) and two microRNA genes (MIRN194‐1, MIRN215) map within the fragile region.

Replication timing of two human common fragile sites: FRA1H and FRA2G.

The mammalian chromosomes present specific sites of gaps or breaks, the common fragile sites (CFSs), when the cells are exposed to DNA replication stress or to some DNA binding compounds. CFSs span hundreds or thousands of kilobases. The analysis of these sequences has not definitively clarified the causes of their fragility. There is considerable evidence that CFSs are regions of late or slowed replication in the presence of sequence elements that have the propensity to form secondary structures, and that the cytogenetic expression of CFSs may be due to unreplicated DNA. In order to analyse the relationship between DNA replication time and fragility, in this work we have investigated the timing of replication of sequences mapping within two CFSs (FRA1H and FRA2G), of syntenic non-fragile sequences and of early and late replicating control sequences by using fluorescent in situ hybridization on interphase nuclei, conventional fluorescence microscopy and confocal microscopy. Our results indicate that the fragile sequences are slow replicating and that they enter G2 phase unreplicated with very high frequency. Thus these regions could sometimes reach mitosis unreplicated or undercondensed and be expressed as chromosome gaps/breakages.

Identification and characterization of U1 small nuclear RNA genes from two crustacean isopod species

Four different units containing three variants of the U1 snRNA gene have been identified in the genome of Asellus aquaticus and only one unit has been identified in the genome of Proasellus coxalis. All four identified U1 snRNA genes can be folded according to the proper secondary structure and possess the functionally useful conserved sequences. Moreover, in the 3′ flanking regions, all genes present both the 3′ box, a conserved sequence required for 3′ processing of mature snRNA, and a polyadenylation signal which is unusual for these genes. The PCR products were used as probes in fluorescent in-situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus and P. coxalis.

Breakages at common fragile sites set boundaries of amplified regions in two leukemia cell lines K562 - Molecular characterization of FRA2H and localization of a new CFS FRA2S.

Genome amplification is often observed in human tumors. The breakage-fusion-bridge (BFB) cycle is the mechanism that often underlies duplicated regions. Some research has indicated common fragile sites (CFS) as possible sites of chromosome breakages at the origin of BFB cycles. Here we searched two human genome regions known as amplification hot spots for any DNA copy number amplifications by analyzing 21 cancer cell lines to investigate the relationship between genomic fragility and amplification. We identified a duplicated region on a chromosomes der(2) present in the karyotype of two analysed leukemia cell lines K562. The two duplicated regions are organized into large palindromes, which suggests that one BFB cycle has occurred. Our findings show that the three breakpoints are localized in the sequence of three CFSs: FRA2H (2q32.1-q32.2), which here has been characterized molecularly; FRA2S (2q22.3-q23.3), a newly localized aphidicolin inducible CFS; and FRA2G (2q24.3-q31).

Characterization of FRA7B, a human common fragile site mapped at the 7p chromosome terminal region

Common fragile sites (CFS) are specific regions of the mammalian chromosomes that are particularly prone to gaps and breaks. They are a cause of genome instability, and the location of many CFS correlates with breakpoints of aberrations recurrent in some cancers. The molecular characterization of some CFS has not clarified the causes of their fragility. In this work, by using fluorescence in situ hybridization analysis with BAC and PAC clones, we determined the DNA sequence of the CFS FRA7B. The FRA7B sequence was then analyzed to identify coding sequences and some structural features possibly involved in fragility. FRA7B spans about 12.2 megabases, and is therefore one of the largest CFS analyzed. It maps at the 7p21.3-22.3 chromosome bands, therefore at the interface of G- and R-band regions that are probably difficult to replicate. A 90-kilobase long sequence that presents very high flexibility values was identified at the very beginning of the more fragile CFS region. Three large genes (THSD7A, SDK1, and MAD1L1) and two miRNA genes (MIRN589 and MIRN339) map in the fragile region. The chromosome band 7p22 is a recurrent breakpoint in chromosome abnormalities in different types of neoplasm. FRA7B is the first characterized CFS located in a chromosome terminal region.

Clonal inheritance of rRNA gene activity: Cytological evidence in human cells

Clone-specific silver staining patterns of the nucleolus organizers (NOs) were observed in clones deriving from a human diploid fibroblast culture. Each specific staining pattern corresponds to one of the nine patterns observed in the general population. Since silver stainability of the NOs is related to rRNA gene function, these data demonstrate the clonal inheritance of rRNA gene activity.