Elena Raimondi

Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

Differences in the organization and chromosomal allocation of satellite DNA between the European long tailed house mice Mus domesticus and Mus musculus

We compared the organization of satellite DNA (stDNA) and its chromosomal allocation inMus domesticus and inMus musculus. The two stDNAs show similar restriction fragment profiles after digestion (probed withM. domesticus stDNA) with some endonucleases of which restriction sequences are present in the 230–240 bp repetitive unit of theM. domesticus stDNA. In contrast, EcoRI digestion reveals thatM. musculus stDNA lacks most of the GAATTC restriction sites, particularly at the level of the half-monomer. The chromosome distribution of stDNA (revealed by anM. domesticus stDNA probe) shows different patterns in theM. domesticus andM. musculus karyotypes, with about 60% ofM. domesticus stDNA retained in theM. musculus genome. It is particularly noteworthy that the pericentromeric regions ofM. musculus chromosomes 1 and X are totally devoid ofM. domesticus stDNA sequences. In both groups, the differences in energy transfer between the stDNA-bound fluorochromes Hoechst 33258 and propidium iodide suggest that AT-rich repeated sequences have a much more clustered array in theM. domesticus stDNA, as if they are organized in tandem repeats longer than those ofM. musculus. Considering the data as a whole, it seems likely that the evolutionary paths of the two stDNAs diverged after the generation of the ancestral 230–240 bp stDNA repetitive unit through the amplification, in theM. domesticus genome, of a family repeat which included the EcoRI GAATTC restriction sequence.

A New Basal Subfamily of mariner Elements in Ceratitis rosa and Other Tephritid Flies

Several copies of highly related transposable elements, Crmar2, Almar1, and Asmar1, are described from the genomes of Ceratitis rosa, Anastrepha ludens, and A. suspensa, respectively. One copy from C. rosa, Crmar2.5, contains a full-length, uninterrupted ORF. All the other copies, from the three species contain a long deletion within the putative ORF. The consensus Crmar2 element has features typical of the mariner/Tc1 superfamily of transposable elements. In particular, the Crmar2 consensus encodes a D,D41D motif, a variant of the D,D34D catalytic domain of mariner elements. Phylogenetic analysis of the relationships of these three elements and other members of the mariner/Tc1 superfamily, based on their encoded amino acid sequences, suggests that they form a new basal subfamily of mariner elements, the rosa subfamily. BLAST analyses identified sequences from other diptera, including Drosophila melanogaster, which appear to be members of the rosa subfamily of mariner elements. Analyses of their molecular evolution suggests that Crmar2 entered the genome of C. rosa in the recent past, a consequence of horizontal transfer.

Two extended arrays of a satellite DNA sequence at the centromere and at the short-arm telomere of Chinese hamster chromosome 5

We have cloned a Chinese hamster chromosome-specific repeated sequence (SatCH5). This satellite is composed of a 33-bp unit organized in two extended tandem arrays. It is localized at the centromere and at the short-arm subtelomere of chromosome 5. Altogether, SatCH5 covers about 1–2 Mb per diploid genome and is not present in other species, including the Syrian hamster and mouse. Since it is known in the Chinese hamster and numerous other vertebrate species that telomeric (TTAGGG)n repeats are localized at the centromeres of several chromosomes, we studied the localization of SatCH5 relative to (TTAGGG)n sequences. Using two-color fluorescence in situ hybridization on stretched chromosomes and on DNA fibers, we have shown that at the centromere of chromosome 5 SatCH5 and the (TTAGGG)n arrays are contiguous. SatCH5 is the first chromosome-specific repetitive sequence located at both the pericentromeric and subtelomeric regions of the same chromosome.

Sister chromatid exchange and proliferation pattern in stimulated lymphocytes of cutaneous malignant melanoma patients

Sister chromatid exchange (SCE) and the proliferative pattern of phytohemagglutinin-stimulated lymphocytes were examined in 36 nonfamilial cutaneous malignant melanoma (CMM) patients. One close relative of each of 27 CMM patients was also examined. All the patients had undergone surgical treatment for the neoplasm, but had received no chemotherapy or radiotherapy. The SCE rates were found to be higher and more variable in a significant fraction of CMM patients, and in relatively fewer unaffected relatives, which is in contrast to findings in unrelated subjects taken as controls. Also, variable and higher proportions of cells in metaphase of the first cell cycle (M1), after 72-hr culture in the presence of bromodeoxyuridine, were more often found among the CMM patients than in the controls; however, no effect of clinical progression of the neoplastic disease on SCE rates or on the lymphoproliferative pattern was observed. The present study indicates heterogeneity among subjects who develop CMM and suggests that the peculiarities of SCE rates and of the lymphoproliferative patterns observed in some of the CMM patients and in a few of their close relatives may be connected with the mechanism of onset of the neoplasm.

Evolution of different subfamilies of mariner elements within the medfly genome inferred from abundance and chromosomal distribution

Abstract.The abundance and distribution pattern of eight mariner elements from three different subfamilies in the genome of the medfly Ceratitis capitata were determined. The copy numbers, as determined by slot-blot analysis, were very different for these elements. Their abundance did not change significantly within the native, the ancient or the newly derived populations, indicating that the rapid colonization process of the medfly had not affected the copy number of mariner elements. The distribution of the mariner elements was analyzed using fluorescent in situ hybridization (FISH) with charge-coupled device (CCD) camera analysis. The pattern of distribution in euchromatin and heterochromatin varied greatly and was distinctive and specific for each element. The implications of these findings are discussed and it is concluded that they generally support the hypothesis of a transposition/selection model in which the abundance and distribution patterns of these elements are regulated primarily by selection against deleterious effects due to meiotic ectopic recombination, while genetic drift would have played a minor role.

Cloning of the bovine prion-like Shadoo (SPRN) gene by comparative analysis of the predicted genomic locus

SPRN is a new prion-like gene coding for Sho, a protein with significant similarity to PrP. SPRN was initially described in zebrafish; however, the strong evolutionary conservation led to the hypothesis that SPRN might be the ancestral prion-like gene. We mapped SPRN in Bos taurus by comparative analysis of the locus and of the predicted flanking genes. BACs, spanning the whole SPRN genomic locus, were assigned to BTA26q23 by radiation hybrid mapping and fluorescent in situ hybridization (FISH). Sequencing of five genes flanking SPRN, namely, ECHS1, PAOX, MTG1, SPRN, and CYP2E1, high-resolution FISH on mechanically stretched chromosomes, and combed BAC DNA allowed us to establish their order and reciprocal orientation. The results confirmed that BTA26q23 corresponds to HSA10q24.3–26.3, which is the site where the human SPRN is located. The gene order in Bos taurus is the same as in man, cen-ECHS1-PAOX-MTG1-SPRN-CYP2E1-tel, but PAOX has a different orientation in the two species. SPRN has the typical two-exon PRNP arrangement, with the CDS fully contained within exon 2; furthermore, it codes for a 143-amino-acid protein with 74.8% identity and 84.7% similarity with the human PRNP. RT-PCR and Northern blot analysis showed that SPRN is expressed at high levels in brain and less in testis and lung.

Insertion of a loxP site in a size-reduced human accessory chromosome

The generation in vitro of mammalian artificial chromosomes, in view of the possibility of developing new technologies for gene therapy, is still an ambitious goal. Mammalian artificial chromosomes, to be used as cloning and expression vectors, have been constructed either by de novo synthesis or by reduction of pre-existing chromosomes. In the work here reported, we introduced a loxP sequence into the pericentromeric region of a chromosome 9-derived X-ray-reduced minichromosome, with the purpose of generating a human chromosome vector (HCV). The modified accessory chromosome is linear and mitotically stable, has lost at least 1400 kb of alpha satellite DNA and normally binds CENP-B, CENP-C and CENP-E. The efficiency of gene targeting via loxP mediated homologous recombination was tested using the histone H2B-Green Fluorescent Protein chimaeric gene as a reporter. The frequency of site-specific insertion of the exogenous sequence was found to be about 50% and to occur in a controlled way with regard to the number of copies. The expression level of the fusion protein was stable over prolonged time in culture.

The origin of a morphologically unidentifiable human supernumerary minichromosome traced through sorting, molecular cloning, and in situ hybridisation.

A supernumerary minichromosome has been detected in a severely malformed patient. Attempts at identifying the marker by conventional approaches were unsuccessful. The physical isolation of the minichromosome by fluorescence activated sorting, molecular cloning of its DNA, and in situ hybridisation experiments performed with single copy DNA probes allowed us to show that it was derived from a rearrangement involving the centromere and the proximal region of the short arm of chromosome 9.

Isolation and characterization of a family of sequences dispersed on the human X chromosome

During a systematic search for X-specific sequences we isolated a DNA fragment (called G1.3) that hybridizes to six further homologous X-specific genomic fragments that map to at least four different regions of the human X chromosome. Genomic segments of 11-30 kb (called G1.3 a, b, c, d, and e or DNF22S1 to DNF22S5) have been subsequently cloned for five of the seven repetitions and characterized by restriction mapping. Single-copy sequences have been used to analyze homology between cloned repetitions, to confirm X specificity, and to regionally localize the repetitions. Sequence homology between members of this family seems to be very high (80-90%) and to extend over at least 5 to 12 kb. In situ hybridization and Southern blotting experiments with a panel of human-rodent hybrid cell lines demonstrated that four of the cloned sequences map to three different regions within Xp21.2-pter and the fifth one (G1.3c) maps to Xq28. The family is present with the same complexity and X specificity in macaques (20-30 x 10(6) years divergence with man), whereas no related sequences were detected in the mouse. To our knowledge small families of dispersed chromosome-specific sequences have been described only for the human Y chromosome. The possible functional or evolutionary significance of this family is discussed.