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Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.

Interstitial telomeric sequences (ITSs) are stretches of telomeric-like repeats located at internal chromosomal sites. We previously demonstrated that ITSs have been inserted during the repair of DNA double-strand breaks in the course of evolution and that some rodent ITSs, called TERC-ITSs, are flanked by fragments retrotranscribed from the telomerase RNA component (TERC). In this work, we carried out an extensive search of TERC-ITSs in 30 vertebrate genomes and identified 41 such loci in 22 species, including in humans and other primates. The fragment retrotranscribed from the TERC RNA varies in different lineages and its sequence seems to be related to the organization of TERC. Through comparative analysis of TERC-ITSs with orthologous empty loci, we demonstrated that, at each locus, the TERC-like sequence and the ITS have been inserted in one step in the course of evolution. Our findings suggest that telomerase participated in a peculiar pathway of DNA double-strand break repair involving retrotranscription of its RNA component and that this mechanism may be active in all vertebrate species. These results add new evidence to the hypothesis that RNA-templated DNA repair mechanisms are active in vertebrate cells.

Interstitial telomeric sequences (ITSs) are short stretches of telomeric-like repeats (TTAGGG)n at nonterminal chromosomal sites. We previously demonstrated that, in the genomes of primates and rodents, ITSs were inserted during the repair of DNA double-strand breaks. These conclusions were derived from sequence comparisons of ITS-containing loci and ITS-less orthologous loci in different species. To our knowledge, insertion polymorphism of ITSs, i.e., the presence of an ITS-containing allele and an ITS-less allele in the same species, has not been described. In this work, we carried out a genome-wide analysis of 2504 human genomic sequences retrieved from the 1000 Genomes Project and a PCR-based analysis of 209 human DNA samples. In spite of the large number of individual genomes analyzed we did not find any evidence of insertion polymorphism in the human population. On the contrary, the analysis of ITS loci in the genome of a single horse individual, the reference genome, allowed us to identify five heterozygous ITS loci, suggesting that insertion polymorphism of ITSs is an important source of genetic variability in this species. Finally, following a comparative sequence analysis of horse ITSs and of their orthologous empty loci in other Perissodactyla, we propose models for the mechanism of ITS insertion during the evolution of this order.

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries.

Individuals differ in realized fitness but the genetic/phenotypic traits that underpin such variation are often unknown. Telomere dynamics may be a major source of variation in fitness traits because physiological telomere shortening depends on environmental and genetic factors and may impair individual performance. Here, we showed that, in a population of a socially monogamous, biparental passerine bird, the barn swallow (Hirundo rustica), breeding in northern Italy, telomere length (TL) of both adult males and females positively correlated with seasonal reproductive and fledging success, as expected because long telomeres are supposed to boost performance. Telomere length was correlated with sexually dimorphic coloration in both sexes, showing for the first time in any species that coloration reliably reflects TL and may mediate mutual mate choice, leading to the observed positive assortative mating for TL in the barn swallow. Thus, TL appears to be associated with variation in a major fitness trait and may be an ultimate target of mate choice, as individuals of both sexes can use coloration to adaptively choose high-quality mates that possess long telomeres.

The typical vertebrate centromeres contain long stretches of highly repeated DNA sequences (satellite DNA). We previously demonstrated that the karyotypes of the species belonging to the genus Equus are characterized by the presence of satellite-free and satellite-based centromeres and represent a unique biological model for the study of centromere organization and behavior. Using horse primary fibroblasts cultured in vitro, we compared the segregation fidelity of chromosome 11, whose centromere is satellite-free, with that of chromosome 13, which has similar size and a centromere containing long stretches of satellite DNA. The mitotic stability of the two chromosomes was compared under normal conditions and under mitotic stress induced by the spindle inhibitor, nocodazole. Two independent molecular-cytogenetic approaches were used—the interphase aneuploidy analysis and the cytokinesis-block micronucleus assay. Both assays were coupled to fluorescence in situ hybridization with chromosome specific probes in order to identify chromosome 11 and chromosome 13, respectively. In addition, we tested if the lack of centromeric satellite DNA affected chromatid cohesion under normal and stress conditions. We demonstrated that, in our system, the segregation fidelity of a chromosome is not influenced by the presence of long stretches of tandem repeats at its centromere. To our knowledge, the present study is the first analysis of the mitotic behavior of a natural satellite-free centromere.

The centromere directs the segregation of chromosomes during mitosis and meiosis. It is a distinct genetic locus whose identity is established through epigenetic mechanisms that depend on the deposition of centromere-specific centromere protein A (CENP-A) nucleosomes. This important chromatin domain has so far escaped comprehensive molecular analysis due to its typical association with highly repetitive satellite DNA. In previous work, we discovered that the centromere of horse chromosome 11 is completely devoid of satellite DNA; this peculiar feature makes it a unique model to dissect the molecular architecture of mammalian centromeres. Here, we exploited this native satellite-free centromere to determine the precise localization of its functional domains in five individuals: We hybridized DNA purified from chromatin immunoprecipitated with an anti CENP-A antibody to a high resolution array (ChIP-on-chip) of the region containing the primary constriction of horse chromosome 11. Strikingly, each individual exhibited a different arrangement of CENP-A binding domains. We then analysed the organization of each domain using a single nucleotide polymorphism (SNP)-based approach and single molecule analysis on chromatin fibres. Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct ‘positional alleles’, each one extending for about 80–160 kb, were found across a region of about 500 kb. Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position. We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles. Electronic supplementary material The online version of this article (doi:10.1007/s00412-014-0493-6) contains supplementary material, which is available to authorized users.

Centromeres are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats called satellite DNA. Here, we describe the isolation and characterization of a 137-bp-long new satellite DNA sequence from the horse genome (EC137), which is also present, even if less abundant, in the domestic donkey, the Grevy's zebra and the Burchelli's zebra. We investigated the chromosomal distribution of the EC137 sequence in these 4 species. Moreover, we analyzed its architectural organization by high-resolution FISH. The position of this sequence with respect to the primary constriction and in relation to the 2 major horse satellite tandem repeats (37cen and 2PI) on horse chromosomes suggests that the new centromeric equine satellite is an accessory DNA element, presumably contributing to the organization of pericentromeric chromatin. FISH on combed DNA fibers reveals that the EC137 satellite is organized in relatively short stretches (2-8 kb) which are strictly intermingled within 37cen or 2PI arrays. This arrangement suggests that interchanges between satellite families are a frequent occurrence in the horse genome.

Archaeological and genetic evidence concerning the time and mode of wild horse (Equus ferus) domestication is still debated. High levels of genetic diversity in horse mtDNA have been detected when analyzing the control region; recurrent mutations, however, tend to blur the structure of the phylogenetic tree. Here, we brought the horse mtDNA phylogeny to the highest level of molecular resolution by analyzing 83 mitochondrial genomes from modern horses across Asia, Europe, the Middle East, and the Americas. Our data reveal 18 major haplogroups (A-R) with radiation times that are mostly confined to the Neolithic and later periods and place the root of the phylogeny corresponding to the Ancestral Mare Mitogenome at ~130-160 thousand years ago. All haplogroups were detected in modern horses from Asia, but F was only found in E. przewalskii--the only remaining wild horse. Therefore, a wide range of matrilineal lineages from the extinct E. ferus underwent domestication in the Eurasian steppes during the Eneolithic period and were transmitted to modern E. caballus breeds. Importantly, now that the major horse haplogroups have been defined, each with diagnostic mutational motifs (in both the coding and control regions), these haplotypes could be easily used to (i) classify well-preserved ancient remains, (ii) (re)assess the haplogroup variation of modern breeds, including Thoroughbreds, and (iii) evaluate the possible role of mtDNA backgrounds in racehorse performance. This research received support from the Italian Ministry of Education, University and Research: Fondo per gli Investimenti della Ricerca di Base (FIRB) Futuro in Ricerca 2008 (to A.A. and A.O.), Progetti Ricerca Interesse Nazionale 2008 (to E.G.) and 2009 (to A.A., O.S., and A.T.); L'ORÉAL Italia per le Donne e la Scienza (to A.O.), Fundação para a Ciência e Tecnologia (FCT), Grant SFRH/BPD/64233/2009 (to P.S.); Consorzio Interuniversitario di Biotecnologie (to M. Santagostino); Fondazione Alma Mater Ticinensis (O.S. and A.T.); and Progetto Ricerca e INNOVAzione nelle attività di miglioramento GENetico animale (INNOVAGEN), Italian Ministry of Agriculture (M. Silvestrelli). Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP) is an Associate Laboratory of the Portuguese Ministry of Science, Technology, and Higher Education and is partially supported by FCT.

Chantier qualité GA; International audience; We mapped six genes (EIF4G3, HSP90, RBBP6, IL8, TERT, and TERC) on the chromosomes of Equus caballus, Equus asinus, Equus grevyi, and Equus burchelli by fluorescence in situ hybridization. Our results add six type I markers to the cytogenetic map of these species and provide new information on the comparative genomics of the genus Equus.

In addition to their location at terminal positions, telomeric-like repeats are also present at internal sites of the chromosomes (intrachromosomal or interstitial telomeric sequences, ITSs). According to their sequence organization and genomic location, two different kinds of ITSs can be identified: (1) heterochromatic ITSs (het-ITSs), large (up to hundreds of kb) stretches of telomeric-like DNA localized mainly at centromeres, and (2) short ITSs (s-ITSs), short stretches of telomeric hexamers distributed at internal sites of the chromosomes. Het-ITSs have been only described in some vertebrate species and they probably represent the remnants of evolutionary chromosomal rearrangements. On the contrary, s-ITSs are probably present in all mammalian genomes although they have been described in detail only in some completely sequenced genomes. Sequence database analysis revealed the presence of 83, 79, 244 and 250 such s-ITSs in the human, chimpanzee, mouse and rat genomes, respectively. Analysis of the flanking sequences suggested that s-ITSs were inserted during the repair of DNA double-strand breaks that occurred in the course of evolution. An extensive comparative analysis of the s-ITS loci and their orthologous ‘empty’ loci confirmed this hypothesis and suggested that the repair event involved the direct action of telomerase. Whereas het-ITSs seem to be intrinsically prone to breakage, the instability of s-ITSs is more controversial. This observation is consistent with the hypothesis that s-ITSs are probably not themselves prone to breakage but represent ‘scars’ of ancient breakage that may have occurred within fragile regions. This study will review the current knowledge on these two types of ITS, their molecular organization, how they arose during evolution, their implications for chromosomal instability and their potential applications as phylogenetic/forensic markers.

Background In mammals, an important source of genomic variation is insertion polymorphism of retrotransposons. These may acquire a functional role when inserted inside genes or in their proximity. The aim of this work was to carry out a genome wide analysis of ERE1 retrotransposons in the horse and to analyze insertion polymorphism in relation to evolution and function. The effect of an ERE1 insertion in the promoter of the myostatin gene, which is involved in muscle development, was also investigated. Results In the horse population, the fraction of ERE1 polymorphic loci is related to the degree of similarity to their consensus sequence. Through the analysis of ERE1 conservation in seven equid species, we established that the level of identity to their consensus is indicative of evolutionary age of insertion. The position of ERE1s relative to genes suggests that some elements have acquired a functional role. Reporter gene assays showed that the ERE1 insertion within the horse myostatin promoter affects gene expression. The frequency of this variant promoter correlates with sport aptitude and racing performance. Conclusions Sequence conservation and insertion polymorphism of ERE1 elements are related to the time of their appearance in the horse lineage, therefore, ERE1s are a useful tool for evolutionary and population studies. Our results suggest that the ERE1 insertion at the myostatin locus has been unwittingly selected by breeders to obtain horses with specific racing abilities. Although a complex combination of environmental and genetic factors contributes to athletic performance, breeding schemes may take into account ERE1 insertion polymorphism at the myostatin promoter. Electronic supplementary material The online version of this article (doi:10.1186/s12863-015-0281-1) contains supplementary material, which is available to authorized users.

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli’s zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years).These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation. D 2005 Elsevier Inc. All rights reserved.

Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli’s zebra and Grevy’s zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

In the karyotype of Equus asinus (domestic donkey, 2n = 62), non-centromeric heterochromatic bands have been described in subcentromeric and telomeric positions. In particular, chromosome 1 is characterised by heterochromatic bands in the proximal region of the long arm and in the short arm; it has been shown that these regions are polymorphic in size. Here we investigated the variation in the intensity and distribution of fluorescence signals observed on donkey chromosome 1 after in situ hybridization with two DNA probes containing fragments from the two major equine satellite DNA families. Our results show that, in Equus asinus chromosome 1, the amount and distribution of large clusters of satellite DNA can define at least nine polymorphic variants of the constitutive heterochromatin that cannot be detected by C-banding alone.

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., Author Summary Centromeres are the functional elements controlling chromosome segregation during cell division. Vertebrate centromeres, which typically contain large amounts of tandem repeats (satellite DNA), are highly conserved for function but not for DNA sequence, suggesting that centromeric function is mainly determined by epigenetic factors. Evolutionary centromere repositioning is the shift of a centromere to a new position in the absence of structural chromosome rearrangements. In previous work, we demonstrated that centromere repositioning was exceptionally frequent during the evolution of the genus Equus (horses, asses, and zebras). In the present paper, we show that several Equus centromeres, including all the previously described evolutionary new centromeres, are apparently satellite-free, supporting the idea that large blocks of repeats are not necessarily required for the stability of centromeres. Our results suggest that centromere repositioning might be a two-step event: first, a neocentromere arises in a satellite-less region; satellite repeats may then colonize this repositioned centromere at a later stage, giving rise to a “mature” centromere. The rapidly evolving Equus species gave us the opportunity to catch snapshots of several evolutionary novel centromeres in different stages during their maturation.

The quantitative variation of a conserved region of the LINE-1 ORF2 sequence was determined in eight species and subspecies of the subgenus Mus (M. m. domesticus, M. m. musculus, M. m. castaneus, M. spicilegus, M. spretus, M. cervicolor, M. cookii, M. caroli) and five Robertsonian races of M. m. domesticus. No differences in LINE-1 ORF2 content were found between all acrocentric or Robertsonian chromosome races, whereas the quantitative variation of the LINE-1 ORF2 sequences detected among the eight taxa partly matches with the clades into which the subgenus is divided. An accumulation of LINE-1 ORF2 elements likely occurred during the evolution of the subgenus. Within the Asiatic clade, M. cervicolor, cookii, and caroli show a low quantity of LINE-1 sequences, also detected within the Palearctic clade in M. m. castaneus and M. spretus, representing perhaps the ancestral condition within the subgenus. On the other hand, M. m. domesticus, M. m. musculus and M. spicilegus showed a high content of LINE-1 ORF2 sequences. Comparison between the chromosomal hybridization pattern of M. m. domesticus, which possesses the highest content, and M. spicilegus did not show any difference in the LINE-1 ORF2 distribution, suggesting that the quantitative variation of this sequence family did not involve chromosome restructuring or a preferential chromosome accumulation, during the evolution of M. m. domesticus.

Telomeres are conserved DNA-protein structures at the termini of eukaryotic chromosomes which contribute to maintenance of genome integrity, and their shortening leads to cell senescence, with negative consequences for organismal functions. Because telomere erosion is influenced by extrinsic and endogenous factors, telomere dynamics may provide a mechanistic basis for evolutionary and physiological trade-offs. Yet, knowledge of fundamental aspects of telomere biology under natural selection regimes, including sex- and context-dependent variation in early-life, and the covariation between telomere dynamics and growth, is scant. In this study of barn swallows (Hirundo rustica) we investigated the sex-dependent telomere erosion during nestling period, and the covariation between relative telomere length and body and plumage growth. Finally, we tested whether any covariation between growth traits and relative telomere length depends on the social environment, as influenced by sibling sex ratio. Relative telomere length declined on average over the period of nestling maximal growth rate (between 7 and 16 days of age) and differently covaried with initial relative telomere length in either sex. The frequency distribution of changes in relative telomere length was bimodal, with most nestlings decreasing and some increasing relative telomere length, but none of the offspring traits predicted the a posteriori identified group to which individual nestlings belonged. Tail and wing length increased with relative telomere length, but more steeply in males than females, and this relationship held both at the within- and among-broods levels. Moreover, the increase in plumage phenotypic values was steeper when the sex ratio of an individual’s siblings was female-biased. Our study provides evidence for telomere shortening during early life according to subtly different dynamics in either sex. Furthermore, it shows that the positive covariation between growth and relative telomere length depends on sex as well as social environment, in terms of sibling sex ratio.

Short blocks of telomeric-like DNA (Interstitial Telomeric Sequences, ITSs) are found far from chromosome ends. We addressed the question as to how such sequences arise by comparing the loci of 10 human ITSs with their genomic orthologs in 12 primate species. The ITSs did not derive from expansion of pre-existing TTAGGG units, as described for other microsatellites, but appeared suddenly during evolution. Nine insertion events were dated along the primate evolutionary tree, the dates ranging between 40 and 6 million years ago. Sequence comparisons suggest that in each case the block of (TTAGGG)n DNA arose as a result of double-strand break repair. In fact, ancestral sequences were either interrupted precisely by the tract of telomeric-like repeats or showed the typical modifications observed at double-strand break repair sites such as short deletions, addition of random sequences, or duplications. Similar conclusions were drawn from the analysis of a chimpanzee-specific ITS. We propose that telomeric sequences were inserted by the capture of a telomeric DNA fragment at the break site or by the telomerase enzyme. Our conclusions indicate that human ITSs are relics of ancient breakage rather than fragile sites themselves, as previously suggested.

The intrachromosomal location of (T2AG3)n telomeric sequences has been reported in several species. It was proposed that interstitial telomeres (ITs) originated through telomeric fusion of ancestral chromosomes. However, the data so far obtained derive mainly from cytogenetic observations. Cloning and database searching of human IT sequences allowed us to identify three classes: (i) short ITs, composed of few, essentially exact T2AG3 units; (ii) subtelomeric ITs, composed of larger arrays (several hundred base pairs) including many degenerate units within subtelomeric domains; (iii) fusion ITs, in which two extended stretches of telomeric repeats are oriented head-to-head. The number of short ITs is over 50 and subtelomeric ITs are probably present at all chromosomal ends. Surprisingly, the telomeric sequence in 2q13 remains the only fusion IT so far characterized, and evidence presented here suggests that another member of this class may be present in 1q41. Different molecular mechanisms generated the three classes. In particular, several short ITs interrupt precisely repetitive elements or are flanked by direct repeats of 10–41 bp, and are conserved in gorilla and chimpanzee. These features strongly suggest that telomeric repeats were inserted at intrachromosomal sites through the repair of double-strand breaks that occurred in the germline during evolution.

Two gastric cancer cell lines, AKG and GK2, were established from a pleural and an ascitic effusion, respectively. GK2 cells have a pseudodiploid karyotype with an add(6)(q27) chromosome in all metaphases examined. The karyotype of AKG cells is highly rearranged: FISH analysis with painting probes has shown that DNA sequences derived from single chromosomes are scattered on several (as many as eight) markers. In this cell line, the C-MYC and the K-RAS oncogenes are amplified. The organization and the copy number of the C-MYC-amplified units are different from the K-RAS units, suggesting that the two oncogenes were amplified independently. The presence of a few marker chromosomes carrying both C-MYC and K-RAS could be due to translocation events that followed the amplification.

A comparative analysis of two primate and two rodent genomes suggests that telomerase was utilized, in some instances, for the repair of DNA double-strand breaks during mammalian evolution., Background In vertebrates, tandem arrays of TTAGGG hexamers are present at both telomeres and intrachromosomal sites (interstitial telomeric sequences (ITSs)). We previously showed that, in primates, ITSs were inserted during the repair of DNA double-strand breaks and proposed that they could arise from either the capture of telomeric fragments or the action of telomerase. Results An extensive comparative analysis of two primate (Homo sapiens and Pan troglodytes) and two rodent (Mus musculus and Rattus norvegicus) genomes allowed us to describe organization and insertion mechanisms of all the informative ITSs present in the four species. Two novel observations support the hypothesis of telomerase involvement in ITS insertion: in a highly significant fraction of informative loci, the ITSs were introduced at break sites where a few nucleotides homologous to the telomeric hexamer were exposed; in the rodent genomes, complex ITS loci are present in which a retrotranscribed fragment of the telomerase RNA, far away from the canonical template, was inserted together with the telomeric repeats. Moreover, mutational analysis of the TTAGGG arrays in the different species suggests that they were inserted as exact telomeric hexamers, further supporting the participation of telomerase in ITS formation. Conclusion These results strongly suggest that telomerase was utilized, in some instances, for the repair of DNA double-strand breaks occurring in the genomes of rodents and primates during evolution. The presence, in the rodent genomes, of sequences retrotranscribed from the telomerase RNA strengthens the hypothesis of the origin of telomerase from an ancient retrotransposon.