Carmen Attolini

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.

Molecular organization of internal telomeric sequences in Chinese hamster chromosomes

In Chinese hamster extended blocks of telomeric-like repeats were previously detected by in situ hybridization at the pericentromeric region of most chromosomes and short arrays were localized at several interstitial sites. In this work, we analyzed the molecular organization of internal telomeric sequences (ITs) in the Chinese hamster genome. In genomic transfers hybridized with a telomeric probe, multiple Bal31 insensitive fragments were detected. Most of the fragments ranged in size between less than 1 kb and more than 100 kb and some were polymorphic. Fluorescence in situ hybridization experiments on DNA fibers and on elongated chromosomes showed that the pericentromeric ITs are composed of extensive and essentially continuous arrays of telomeric-like sequences. We then isolated three genomic regions which contain short ITs. These ITs are localized at interstitial sites (3q13-15, 3q21-26, 1p26) and are composed of 29-126 bp of (TTAGGG)(n) repeats. A peculiar feature of all the three ITs is the AT richness of the flanking sequences. Since AT-rich DNA is known to be unstable and characteristic of several mammalian fragile sites, we propose that the three ITs were inserted at these sites during the repair of double strand breaks.

Intrachromosomal telomere-like DNA sequences in Chinese hamster.

The simple repetitive sequence (TTAGGG)n constitutes the telomeres of vertebrate chromosomes. In several species, telomeric repeats have been observed at intrachromosomal sites (Meyne et al. 1990). It has been proposed that interstitial (TrAGGG)n sequences may be prone to fragility and recombination and that breakage within such repetitions may produce telomere-like structures that stabilize the ends of rearranged chromosomes (Ijdo et al. 1991; Meyne et al. 1990). This hypothesis is supported by the cytogenetic correlation between induced breaks and intrachromosomal telomeric sites in Chinese hamster cell lines (Alvarez et al. 1993; Balajee et al. 1994). Furthermore, it has been shown that the recombination events that accompanied CAD gene amplification frequently involved (TTAGGG) repetitions (Bertoni et al. 1994) and that the introduction of human telomeres into rodent or human cells could induce chromosome fragmentation (Farr et al. 1991; Barnett et al. 1993). In Chinese hamster the precise localization of (TTAGGG)n sequences has not been analyzed. In this paper we report the chromosomal localization of these sequences in primary diploid fibroblasts, and we show that they map at telomeric, centromeric and interstitial sites. The interstitial sequences, which have not been described before in Chinese hamster, contain a relatively small number of repeats and may derive from telomere fusion or centromere inactivation of ancestral chromosomes. Moreover, we have observed that spontaneous breaks, occurring during the senescence crisis of primary fibroblasts, are located preferentially within chromosome bands containing (TTAGGG)n repeats. We then mapped the (TTAGGG)n sequences in a CHO-K1 derived cell line and showed that the chromosomes can be identified without banding. New information about the origin of some marker chromosomes has been obtained. Metaphase chromosomes from a Chinese hamster (CricetuIus griseus) primary fibroblast cell line (CHL) were hybridized in situ with a (TTAGGG)n probe and G-banded after hybridization according to the methods previously described (Bertoni et al. 1994). The probe was a mixture of biotinylated synthetic (TTAGGG)n fragments with a size range between 1 and 20 kb. The localization of hybridization signals has been analyzed on 37 metaphases, which were obtained from cells passaged 8 times. In Fig. la two examples from each chromosome pair are shown. The karyotype of CHL cells is identical to the normal Chinese hamster karyotype, which has been described (Ray and Mohandas 1976; Simi et al. 1988). Briefly, 11 chromosome pairs are present: 9 metacentric or submetacentric (1, 2, 3, 4, 8, 9, 10, X, and Y) and 3 acrocentric pairs (5, 6, and 7). All chromosomes show hybridization signals at both ends, indicating that long stretches of (TTAGGG)n repetitions are present at the telomeres. A subterminal localization of the telomeric signal, which is observed in some CHL chromosomes of Fig. la, has been previously described by others and seems to be due to chromosome condensation (Moyzis et al. 1988).

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.

Intrachromosomal telomeric repeats and stabilization of truncated chromosomes in V79 Chinese hamster cells

(TTAGGG)n sequences have been localized on the chromosomes of the Chinese hamster V79 cell line. A correlation between telomeric-like repeats and chromosome breakage has been found. Moreover, the analysis of the truncated chromosomes, typical of this cell line, has suggested that intrachromosomal (TTAGGG)n DNA may be important in the stabilization of the new telomeres.

On the identity of dnaP and dnaF genes of Bacillus subtilis

SummaryThe dnaP strains of Bacillus subtilis are altered in the initiation of DNA replication at high temperature (Riva et al., 1975). Fine mapping of the gene shows that it is located very close to the dnaF gene, described by Karamata and Gross (1970) and mapped by Love et al. (1976) in the polC region. The phenotype of both mutants is indistinguishable: the DNA synthesis stops at non permissive temperature after synthesizing an amount of DNA equivalent to the completion of the rounds of replication already initiated; at permissive temperature they are abnormally sensitive to MMS and are reduced in the ability to be transformed. Both mutants are to be considered as belonging to the dnaF locus.The dnaF gene is very close to the polC gene, which specifies the DNA polymerase III of B. subtilis. The DNA polymerase III of the dnaF mutants is not temperature sensitive in vitro, however, the level of this enzyme is lower by a factor of 4 or 5 in the dnaF mutants, at the permissive temperature. Following shift of dnaF cultures to the non permissive temperature, the level of DNA polymerase III activity specifically decreases further by a factor of at least 10 in the mutant, whereas the DNA polymerase I level is unaffected.The possible roles of the dnaF gene in the control of the cellular level of the DNA polymerase III, and the possibility of a regulatory role of DNA polymerase III in the initiation of DNA replication in bacteria are discussed.

A new mutant of Bacillus subtilis altered in the initiation of chromosome replication

SummaryWe have isolated a new mutant of Bacillus subtilis temperature sensitive in DNA replication; its properties are those of an initiation mutant. When liquid cultures are shifted to 48° DNA replication is the first macromolecular synthesis that stops, but only after synthesis of the amount of DNA predicted for the completion of one replication round. When spores of the mutant are germinated and shifted to 48° at subsequent times, one round of DNA replication is observed only when the shift occurs between 60 and 100 min; earlier shifts do not allow replication to start, later shifts allow more than one replication. The DNA replicated after a shift to high temperature is enriched in markers close to the terminus. The reinitiation of DNA replication stopped by the high temperature, takes place following a shift to a permissive temperature only if protein synthesis is allowed. Examination of DNA replication following toluene treatment shows that the elongation of DNA chains is not affected at the non-permissive temperature.This mutant is shown by PBS-1 mapping to correspond to a new gene denominated dna P, which is located between the thy A and fur A genes and is distinct from all the mapped dna and rec genes of Bacillus subtilis. The mutation confers to the cells also a deficiency in the ability to be transformed, to be transfected with SPP1 phage DNA, and to survive treatment with methyl-methane sulfonate. These deficiencies, observed at the permissive temperature, are no more temperature dependent than in the parental strain. The ability to perform homologous and heterologous transduction with PBS-1 phage and the sensitivity to ultraviolet radiation or mitomycin C are normal.

Assignment of the Equus caballus interleukin 8 gene (IL8) to chromosome 3q14.2→q14.3 by in situ hybridization

Human interleukin 8 (IL-8) is an anti-infl ammatory cytokine. A signifi cant increase of the IL-8 concentration has been found in the plasma of marathon runners (Suzuki et al., 2000) and a similar upregulation of this interleukin was recently detected in lymphocytes of horses immediately after intense exercise (Cappelli, Verini-Supplizi, Capomaccio and Silvestrelli unpublished results). Equus caballus (ECA) IL8 full length cDNA, isolated from peripheral blood lymphocytes of an endurance horse, was sequenced (GenBank accession number AY184956). The mRNA sequence is 1089 nt in length and contains a 306 nt region that putatively codes for a 101 amino acid protein. In the framework of studying the modulation of gene expression profi les in racing horses, we report here the chromosomal location of ECA IL8 . This result adds a new type I marker to the FISH map of the horse.

DNA Repair in Nuclei Isolated from Hela Cells

We have studied the DNA repair synthesis in HeLa cell isolated nuclei in presence of aphidicolin, which selectively inhibits DNA polymerase alpha but not DNA polymerases beta and gamma. The drug strongly depresses the DNA synthesis rate both in vivo and in the subcellular system but seems not to interfere with DNA repair. The enhancement in nucleotide incorporation by isolated nuclei after UV-irradiation is observed only in the presence of ATP.