Human intrachromosomal telomeric-like repeats: sequence organization and mechanisms of origin

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.     

The telomeric region is excluded from nucleosomal fragmentation during apoptosis, but the bulk nuclear chromatin is randomly degraded

Characteristic steps during cellular apoptosis are the induction of chromatin condensation and subsequent DNA fragmentation, finally leading to the formation of oligomers of nucleosomes. We have examined the kinetics and local distribution of this nucleosomal fragmentation within different genomic regions. For the induction of apoptosis, HL60 cells were treated with the water-soluble camptothecin derivative topotecan (a topoisomerase I inhibitor). The genomic origin of the fragments was analysed by Southern blot hybridisation of the cleaved DNA. In these experiments we observed similar hybridisation patterns of the fragmented DNA, indicating a random and synchronous cleavage of the nuclear chromatin. However, hybridisation with a telomeric probe revealed that, in contrast to the other analysed genomic regions, the telomeric chromatin was not cleaved into nucleosomal fragments despite our observation that the telomeric DNA in HL60 cells is organised in nucleosomes. We determined just a minor shortening of the telomeric repeats early during apoptosis. These observations suggest that telomeric chromatin is excluded from internucleosomal cleavage during apoptosis.     

Exploring evolution in Ceboidea (Platyrrhini, primates) by Williams-Beuren probe (HSA 7q11.23) chromosome mapping

The ancestral platyrrhine karyotype was characterised by a syntenic association of human 5 and a small segment of human 7 orthologues. This large syntenic association has undergone numerous rearrangements in various phylogenetic lines. We used a locus-specific molecular cytogenetic approach to study the chromosomal evolution of the human 7q11.23 orthologous sequences (William-Beuren syndrome, WS) in various Ceboidea (Platyrrhini) species. The fluorescent in situ hybridisation of the WS probe revealed a two-way pattern of chromosomal organisation that suggests various evolutionary scenarios. The first pattern (seen in Callimico and Saimiri) includes a fairly simple disruption of the 7/5 syntenic association by a chromosome fission. The second pattern (seen in Atelinae, Alouattinae and in Callicebus) is characterised by an increasing complexity in the 7/5 association as a consequence of a series of inversions and translocations resulting in different syntenic associations. These data support recent proposals for phylogenomic groupings of New World monkeys. The study also illustrates how single-locus probe hybridisations can reveal intrachromosomal rearrangements.     

Biocide gene(s) and biocidal activity in different strains of Bacillus sphaericus. Expression of the gene(s) in E. coli maxicells

Summary The recently cloned biocidal determinant of the highly toxic strain of B. sphaericus 1593M (Ganesan et al. 1983) was used as probe to investigate homologous sequences in different toxic and non-toxic strains of B. sphaericus. It was found that the potent strains we have analysed are characterised by the presence of DNA sequences (6.6, 6.4, 5.8, 1.6, 1.3 and 0.6 Kb) not found in the non-toxic strains. These results further show that one of the two weakly toxic strains analysed presents a hybridisation pattern completely different from that observed with the highly potent strains of B. sphaericus. When the DNA of the two non-toxic strains was analysed, SSII-I failed to hybridise to the probe and Rem4 exhibited mainly one hybridisable sequence of 2.3 Kb not detectable in the toxic strains.No region of homology to the probe was found in the DNA of two strains of B. thuringiensis (var. berliner and var. israeliensis) analysed.By dot blot hybridisation experiments it was estimated that the larvicidal determinant might be present in about one to three copies per genome.With the use of E. coli maxicells we have shown further that the toxin gene(s) encoded four polypeptides with molecular weights of 21, 19, 15, and 12 Kd. The significance of these findings is discussed.     

Fluorescence in situ hybridization of rDNA, telomeric (TTAGGG)n and (GATA)n repeats in the red abalone Haliotis rufescens (Archaeogastropoda: Haliotidae)

The physical location of 18S-5.8S-28S rDNA, telomeric sequences with (TTAGGG)n DNA probe and (GATA)n microsatellites were performed by fluorescence in situ hybridization in chromosomes of red abalone Haliotis rufescens. The karyotype of red abalone showed a diploid number of 36 (8M+9SM+1ST). FISH performed with rDNA probe, showed the location of major ribosomal clusters in the terminal region of the large arms of two submetacentric pairs (chromosome 4 and 5). Localization of heteromorphisms of FISH-rDNA was found between chromosome homologues and sister chromatids in all metaphases analyzed. This indicates that rDNA clusters are variable within the red abalone genome. The variability in the NOR-bearing reported using silver staining in other gastropods and our result are discussed. In addition, the presence of microsatellite (TTAGGG)n and (GATA)n was demonstrated after FISH treatment by DNA probes. The telomeric sequence occurred at the ends of all mitotic chromosomes, while the (GATA)n repetitive was found on chromosomal interstitial zones as well as at the telomeres in abalone chromosomes.     

Evolutionary breakpoints are co-localized with fragile sites and intrachromosomal telomeric sequences in primates

The concentration of evolutionary breakpoints in primate karyotypes in some particular regions or chromosome bands suggests that these chromosome regions are more prone to breakage. This is the first extensive comparative study which investigates a possible relationship of two genetic markers (intrachromosomal telomeric sequences [TTAGGG]n, [ITSs] and fragile sites [FSs]), which are implicated in the evolutionary process as well as in chromosome rearrangements. For this purpose, we have analyzed: (a) the cytogenetic expression of aphidicolin-induced FSs in Cebus apella and Cebus nigrivittatus (F. Cebidae, Platyrrhini) and Mandrillus sphinx (F. Cercopithecidae, Catarrhini), and (b) the intrachromosomal position of telomeric-like sequences by FISH with a synthetic (TTAGGG)n probe in C. apella chromosomes. The multinomial FSM statistical model allowed us to determinate 53 FSs in C. apella, 16 FSs in C. nigrivittatus and 50 FSs in M. sphinx. As expected, all telomeres hybridized with the probe, and 55 intrachromosomal loci were also detected in the Cebus apella karyotype. The chi(2) test indicates that the coincidence of the location of Cebus and Mandrillus FSs with the location of human FSs is significant (P < 0.005). Based on a comparative cytogenetic study among different primate species we have identified (or described) the chromosome bands in the karyotypes of Papionini and Cebus species implicated in evolutionary reorganizations. More than 80% of these evolutionary breakpoints are located in chromosome bands that express FSs and/or contain ITSs.     

Chromosomal localization of the telomeric (TTAGGG)n sequence in eight species of New World Primates (Neotropical Primates, Platyrrhini)

Chromosomal localization of the telomeric sequence (TTAGGG)(n) in eight New World Primates (Platyrrhini) (Alouatta caraya, Alouatta palliata, Alouatta guariba clamitans, Aotus azarae, Ateles chamek, Cebus nigritus, Cebus paraguayanus, and Saimiri boliviensis) using Fluorescence In Situ Hybridization (FISH) with a peptide nucleic acid (PNA) pantelomeric probe and their possible relationship with the C-banding pattern were analyzed. FISH showed telomeric signals only at the terminal regions of chromosomes from all the species analyzed. Although all of them showed centromeric C+ bands and different size and location of extracentromeric C+ bands, none, except Aotus azarae exhibited (peri)centromeric interstitial telomere-like sequences (ITS). The presence of ITS in Aotus azarae was limited to one pair of submetacentric chromosomes and very likely represents telomeric sequences remaining after a fusion event of ancestral chromosomes during karyotype evolution. Therefore, our data indicate that the distribution of heterochromatin blocks do not correlate with the presence of ITS. However, we cannot rule out the possibility that simple ITS arrays with a few copies of the (TTAGGG)(n) sequence, not detectable by conventional FISH, might play a role in the karyotypic evolution of Ceboidea. Further FISH and molecular studies will be needed to confirm this hypothesis.     

Early Expression of a Trypanosoma brucei VSG Gene Duplicated from an Incomplete Basic Copy

Intrachromosomal variant surface glycoprotein (VSG) genes in Trypanosoma brucei are expressed by a mechanism involving gene conversion. The 3'boundary of gene conversion is usually within the last 130 bp of the VSG gene, a region of partially conserved sequences. We report here the loss of the predominant telomeric A VSG gene in the cloned variant antigenic type (VAT) 5^A3, leaving only an intrachromosomal A VSG gene (the A-B gene). The nucleotide sequence of the A-B VSG gene reveals that it lacks the normal VSG 3' sequence. Surprisingly, we find cells expressing this A-B VSG gene in relapse populations arising from VAT 5^A3. Since the A VSG mRNAs from these cells have a normal 3' sequence, the incomplete A-B VSG gene must be expressed via a partial gene conversion that supplies the functional 3'end. Although the A-B VSG gene is no longer predominant like the telomeric A VSG gene, it is still expressed more frequently than other intrachromosomal VSG genes, suggesting that factors other than a telomeric location determine whether a VSG gene is expressed early in a serodeme.     

Chromosomal diversity in three species of electric fish (Apteronotidae,Gymnotiformes) from the Amazon Basin

Cytogenetic studies were carried out on samples of Parapteronotus hasemani, Sternarchogiton preto and Sternarchorhamphus muelleri (Apteronotidae, Gymnotiformes) from the Amazon basin. The first two species exhibited both a 2n = 52 karyotype, but differed in their karyotypic formulae, distribution of constitutive heterochromatin, and chromosomal location of the NOR. The third species, Sternarchorhamphus muelleri, was found to have a 2n = 32 karyotype. In all three species the DAPI and chromomycin A3 staining results were consistent with the C-banding results and nucleolar organizer region (NOR) localization. The 18S rDNA probe confirmed that there was only one pair of ribosomal DNA cistron bearers per species. The telomeric probe did not reveal interstitial telomeric sequences (ITS). The karyotypic differences among these species can be used for taxonomic identification. These data will be useful in future studies of these fishes and help understanding the phylogenetic relationships and chromosomal evolution of the Apteronotidae.     

Chromosomal distribution of interstitial telomeric sequences in nine neotropical primates (Platyrrhini): possible implications in evolution and phylogeny

To localize interstitial telomeric sequences (ITSs) and to test whether their pattern of distribution could be linked to chromosomal evolution, we hybridized telomeric sequence probes (peptide nucleic acid, PNA) on metaphases of New World monkeys: Callithrix argentata, Callithrix jacchus, Cebuella pygmaea, Saguinus oedipus, Saimiri sciureus, Aotus lemurinus griseimembra, Aotus nancymaae (Cebidae), Lagothrix lagotricha (Atelidae) and Callicebus moloch (Pithecidae), characterized by a rapid radiation and a high rate of chromosomal rearrangements. Our analysis of the probe signal localization allowed us to show in all the species analysed, as normally, the telomeric location at the terminal ends of chromosomes and unexpected signal distributions in some species. Indeed, in three species among the nine studied, Aotus lemurinus griseimembra, Aotus nancymaae (Cebidae) and Lagothrix lagotricha (Atelidae), we showed a high variability in terms of localization and degree of amplification of interstitial telomeric sequences, especially for the ones found at centromeric or pericentromeric positions (het‐ITS). A comparative analysis, between species, of homologous chromosomes to human syntenies, on which we have found positive interspersed PNA signals, allowed us to explain the observed pattern of ITS distribution as results of chromosomal rearrangements in the neotropical primates analysed. This evidence permitted us to discuss the possible implication of ITSs as phylogenetic markers for closely related species. Moreover, reviewing previous literature data of ITSs distribution in Primates and in the light of our results, we suggest an underestimation of ITSs and highlight the importance of the molecular cytogenetics approach in characterizing ITSs, which role is still not clarified.     

Fragile site and interstitial telomere repeat sequences at the fusion point of a de novo (Y;13) translocation

We describe a novel fragile site in a rearranged chromosome, associated with the presence of telomeric repeat sequences at the fusion point of a translocation between chromosomes 13 and Y. The case reported in this study shows a de novo (Y;13) translocation, which appears to represent fusion of an apparently intact chromosome Y with a chromosome 13 that has lost only part of its short arm. Ten percent of the cells show a normal karyotype without the (Y;13) translocation. Molecular cytogenetic studies of the derived Y;13 chromosome revealed three hybridization sites of the telomeric probes – one at each end and one at the breakpoint junction. A fragile site is also observed in the intrachromosomic telomeric region. This coincidence suggests that the telomere repeat sequences (TTAGGG)n, when present at an interstitial chromosomal location, can promote the formation of a novel fragile site. Received: 15 November 1995 / Revised: 6 March 1996     

Oligonucleotide-primed in situ DNA synthesis (PRINS): a method for chromosome mapping, banding, and investigation of sequence organization

Oligonucleotides were annealed to complementary sequences in fixed human metaphase chromosomes and extended with DNA polymerase. The newly synthesized fragments were labeled by incorporating bio-11-dUTP instead of TTP, and the sites of synthesis were detected by immunocytochemistry, using fluorochromes as the reporter molecules. We have obtained clear localization with oligonucleotides from alphoid (centromeric sequences), simple sequence (satellite) DNAs, a variety of Alu-dispersed repeated sequences, and oligonucleotides derived from the Tetrahymena and Trypanosoma telomere-specific sequences. The simple sequence and alphoid oligonucleotides gave results at least comparable to those obtained using the whole molecule as a probe for in situ hybridization, whereas the Alu oligonucleotides produced a diversity of results which depended on the absolute length and location of the oligonucleotide within the Alu sequence. The telomere-specific oligomers also produced a variety of results. The G-rich Trypanosoma oligomer and its complementary C-rich sequence produced strong telomeric signals and some interstitial signals on mouse chromosomes, but only weak telomeric signals on human chromosomes. The G-rich Tetrahymena oligomer produced detectable telomeric signals on human chromosomes. The technique appears to be a valuable extension of present tools for mapping and examining the organization of DNA sequences within chromosomes.     

Distribution of non-telomeric sites of the (TTAGGG)n telomeric sequence in vertebrate chromosomes

The intrachromosomal distribution of non-telomeric sites of the (TTAGGG)_n telomeric repeat was determined for 100 vertebrate species. The most common non-telomeric location of this sequence was in the pericentric regions of chromosomes. A variety of species showed relatively large amounts of this sequence present within regions of constitutive heterochromatin. We discuss possible relationships between the non-telomeric distribution of the (TTAGGG)_n sequence and the process of karyotype evolution, during which these sites may provide potential new telomeres.     

Detection of interstitial telomeric sequences in the Arctic charr (Salvelinus alpinus) (Teleostei: Salmonidae)

Highly polymorphic Arctic charr ( Salvelinus alpinus Linnaeus, 1758) chromosomes were studied using conventional and molecular methods. The diploid chromosome number in the studied individuals was 2n = 81 or 2n = 82, with a fundamental arm number (NF) = 100. These differences are due to Robertsonian fusions. Interindividual variation in the number and size of DAPI and CMA(3) positively stained chromatin sites was observed in studied specimens. In the case of two individuals, the subtelomeric region of the long arm (q) of the largest acrocentric chromosome (chromosome number 10) was positively stained by CMA(3) fluorochrome. Both primed in situ labelling (PRINS) and fluorescence in situ hybridization (FISH) revealed that this CMA(3)-positive region was flanked by telomeric sequences. Previously, the subterminal position of interstitial telomeric sequences located in the vicinity of the CMA(3)-positive guanine-rich chromatin have been described in two other Salvelinus species, brook trout ( Salvelinus fontinalis ) and lake trout ( Salvelinus namaycush ). Moreover, multichromosomal location and variation in size of CMA(3) bands have been observed in various Salvelinus taxa, including fishes with internally located telomeric sequences. These results suggest that relocation of CMA(3)-positive chromatin segments in these species may be facilitated by flanking interstitial telomeric sequences (ITSs).     

Robertsonian metacentrics of the house musk shrew (Suncus murinus, Insectivora, Soricidae) lose the telomeric sequences in the centromeric area

The house musk shrew, Suncus murinus, is polymorphic for five Robertsonian translocations (Rb8.17, 9.13, 10.12, 11.16, 14.15). Fluorescence in situ hybridisation with a biotin-labelled oligonucleotide, (TTAGGG)7, was performed to localise the telomeric DNA sequences at Rb chromosomes of heterozygous shrews. Hybridisation signals were observed at both ends of all chromosomes, but not at the pericentromeric areas of any of the Robertsonian metacentrics. Our results indicate a complete loss of the telomeric sequences at the fusion points of the Rb metacentrics in S. murinus.     

Genomic organization of telomeric and subtelomeric sequences of Leishmania (Leishmania) amazonensis

Telomeres are DNA-protein complexes that protect linear chromosomes from degradation and fusions. Telomeric DNA is repetitive and G-rich, and protrudes towards the end of the chromosomes as 3'G-overhangs. In Leishmania spp., sequences adjacent to telomeres comprise the Leishmania conserved telomere associated sequences (LCTAS) that are around 100 bp long and contain two conserved sequence elements (CSB1 and CSB2), in addition to non-conserved sequences. The aim of this work was to study the genomic organization of Leishmania (Leishmania) amazonensis telomeric/subtelomeric sequences. Leishmania amazonensis chromosomes were separated in a single Pulsed Field Gel Electrophoresis (PFGE) gel as 25 ethidium bromide-stained bands. All of the bands hybridized with the telomeric probe (5'-TTAGGG-3')3 and with probes generated from the conserved subtelomeric elements (CSB1, CSB2). Terminal restriction fragments (TRF) of L. amazonensis chromosomes were analyzed by hybridizing restriction digested genomic DNA and chromosomal DNA separated in 2D-PFGE with the telomeric probe. The L. amazonensis TRF was estimated to be approximately 3.3 kb long and the telomeres were polymorphic and ranged in size from 0.2 to 1.0 kb. Afa I restriction sites within the conserved CSB1 elements released the telomeres from the rest of the chromosome. Bal 31-sensitive analysis confirmed the presence of terminal Afa I restriction sites and served to differentiate telomeric fragments from interstitial internal sequences. The size of the L. amazonensis 3' G-overhang was estimated by non-denaturing Southern blotting to be approximately 12 nt long. Using similar approaches, the subtelomeric domains CSB1 and CSB2 were found to be present in a low copy number compared to telomeres and were organized in blocks of 0.3-1.5 kb flanked by Hinf I and Hae III restriction sites. A model for the organization of L. amazonensis chromosomal ends is provided.     

Comparison of different kinds of probes used for analysis of variant telomeric sequences

In this work we aimed to compare and critically evaluate results obtained by different types of probes used for hybridisation to detect variant telomeric sequences with respect to their reliability and information value. Using slot-blot hybridisation we investigated three types of probes (oligonucleotides, cloned fragments and concatenated probes) under various conditions of hybridisation and washing. The concatenated probes exhibited the highest specificity although all three types are suitable for hybridisation of telomeric sequences under appropriate experimental conditions. We demonstrate how understanding generated from these data enables interpretation of hybridisation patterns of oligonucleotide probes to genomic DNAs.     

The distribution of sequences complementary to human satellite DNAs I,II and IV in the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus)

Human satellite DNAs I, II and IV were transcribed to yield radioactive complementary RNAs (cRNAs). These cRNAs were hybridised to metaphase chromosomes of man, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus). The results of this in situ hybridisation were analysed quantitatively and compared with accepted chromosome homologies based on Giemsa banding patterns. The cRNA to satellite II (cRNAII) did not hybridise to chimpanzee chromosomes, although its hybridisation to chromosomes of gorilla and orang utan yielded more autoradiograph grains than hybridisation to human chromosomes, and cRNAIV hybridised to many chromosomes of gorilla and chimpanzee but was almost entirely restricted to the Y chromosome in orang utan. Most sites of hybridisation were located on homologous chromosomes in all four species, but there were a number of sites which showed no correspondence between satellite DNA location and chromosome banding patterns, and others where a given chromosomal location hybridised with different cRNAs in each species. These results are in contrast to those found for many transcribed DNA sequences, where the same sequence is usually located at homologous chromosome sites in different species, and appear to cast doubt on many proposed models of satellite DNA function.     

Telomeric repeat [TTAGGG]n sequences of human chromosomes are conserved in chimpanzee (Pan troglodytes)

Summary Using a series of genetic parameters, attempts have been made for more than two decades to establish the close kinship of human (Homo sapiens) with chimpanzee (Pan troglodytes). Molecular and cytogenetic data presently suggest that the two species are closely related. The recent isolation of a human telomeric probe (P5097-B.5) has prompted us to cross hybridize it to chimpanzee chromosomes in order to explore convergence and/or divergence of the telomeric repeat sequences (TTAGGG)_n. On hybridization, the human probe bound to both ends (telomeres) of chimpanzee chromosomes, suggesting a concerted evolution of tandemly repeated short simple sequences (TTAGGG)_n. Even the terminal heterochromatin of chimpanzee chromosomes was found to be endowed with telomeric repeats, suggesting that evolution of heterochromatin and capping with tandemly repeated short sequences are highly complex phenomena.     

Location of repetitious DNA in the chromosomes of the desert locust (Schistocerca gregaria)

A modified Giemsa staining technique and the in situ hybridisation technique, have been used to investigate the localisation of highly repeated sequences in the karyotype of the locust Schistocerca gregaria. The centromeric regions are stained densely with Giemsa and further Giemsa-stained bands occur at the telomeric region of the short (S) chromosomes. RNA complementary to repetitious DNA hybridised to loci scattered along the whole length of the chromosomes, with concentrations of grains at the centromeric regions of all the chromosomes and also at the telomeric regions of the S chromosomes.