Distribution of telomeric (TTAGGG)<Subscript>n</Subscript> sequences in avian chromosomes

The physical ends of mammalian and other vertebrate chromosomes consist of tandemly repeated (TTAGGG)(n) hexamers, nucleating a specialized telomeric structure. However, (TTAGGG)(n) sequences can also occur at non-telomeric sites, providing important insights into karyotypic evolution. By fluorescence in situ hybridization (FISH) we studied the chromosomal distribution of (TTAGGG)(n) sequences in 16 bird species, representing seven different orders. Many species, in particular the ratites, display (TTAGGG)(n) hybridization signals in interstitial and centromeric regions of their macrochromosomes in addition to the typical telomeric signals. In some but not all species these non-telomeric sites coincide with C-band-positive heterochromatin. The retention and/or amplification of telomeric (TTAGGG)(n) repeats at interstitial and centromeric sites may indicate the fusion of ancestral chromosomes. Compared with the macrochromosomes, the microchromosomes of most species are enriched with (TTAGGG)(n) sequences, displaying heterogeneous hybridization patterns. We propose that this high density of (TTAGGG)(n) repeats contributes to the exceptionally high meiotic recombination rate of avian microchromosomes.     

FISH-mapping of 18S ribosomal RNA genes and telomeric sequences in the Japanese bitterlings Rhodeus ocellatus kurumeus and Tanakia limbata (Pisces,Cyprinidae) reveals significant cytogenetic differences in morphologically similar karyotypes

The Japanese rose bitterling, Rhodeus ocellatus kurumeus, and the oily bitterling, Tanakia limbata, were cytogenetically studied by silver (Ag)- and chromomycin A₃ (CMA₃)-staining, by C-banding and by mapping of the 18S ribosomal genes and of the (TTAGGG) _n telomeric sequence. These two representative species of related genera of the subfamily Acheilognathinae show very similar chromosome complements. Nevertheless, significant differences in the chromosomal distribution of nucleolus organizer regions (NORs) and interstitial telomeric sequences were observed. Whereas R. ocellatus kurumeus shows a single NOR-bearing chromosome pair, T. limbata is characterized by a higher number of variable NORs. Multiple telomeric sequence sites were found at the pericentromeric regions of several chromosomes in the rose bitterling. No telomeric sequence sites were detected near centromeres, but they were found to be scattered along the NORs in the oily bitterling. Two karyoevolutive trends might have been identified in the subfamily.     

Characterization of the telomere regions of scleractinian coral, <Emphasis Type="Italic">Acropora surculosa</Emphasis>

Terminal ends of vertebrate chromosomes are protected by tandem repeats of the sequence (TTAGGG). First thought to be vertebrate specific, (TTAGGG) _n has recently been identified in several aquatic invertebrates including sea urchin (Strongylocentrotus purpuratus), bay scallop (Argopecten irradians), and wedgeshell clam (Donax trunculus). We analyzed genomic DNA from scleractinian corals, Acropora surculosa, Favia pallida, Leptoria phrygia, and Goniastrea retiformis to determine the telomere sequence. Southern blot analysis suggests the presence of the vertebrate telomere repeats in all four species. Treatment of A. surculosa sperm DNA with Bal31 exonuclease revealed progressive shortening of the DNA fragments positive for the (TTAGGG)₂₂ sequence, supporting location of the repeats at the chromosome ends. The presence of the vertebrate telomere repeats in corals is evidence that the (TTAGGG) _n sequence is highly conserved among a divergent group of vertebrate and invertebrate species. Corals are members of the Lower Metazoans, the group of organisms that span the gap between the fungi and higher metazoans. Corals are the most basal organism reported to have the (TTAGGG) _n sequence to date, which suggests that the vertebrate telomere sequence may be much older than previously thought and that corals may share a number of genes with their higher relatives.     

Chromosomal Location of Ribosomal DNA (rDNA), (GATA)n and (TTAGGG)n Telomeric Repeats in the Neogastropod Fasciolaria Lignaria (Mollusca: Prosobranchia)

This paper reports on a successful application of fluorescent in situhybridization (FISH) with three repetitive DNA probes (ribosomal DNA (rDNA), (GATA)_nand (TTAGGG)_n) in the chromosomes of Fasciolaria lignaria(Mollusca: Prosobranchia: Neogastropoda). rDNA FISH consistently identified four chromosome pairs per spread in the three examined specimens. The telomeric sequence (TTAGGG)_nhybridized with termini of all chromosomes. GATA FISH revealed abundant, dispersed minisatellite regions which were not associated to the XY sex-determining mechanism as indicated by the absence of a Y specific pattern of labelling. This revised version was published online in July 2006 with corrections to the Cover Date.     

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences

Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]₆ sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.     

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences

Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]₆ sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.     

Comparative Molecular Cytogenetic Analysis of Two Congeneric Species, <Emphasis Type="Italic">Mugil Curema</Emphasis> and <Emphasis Type="Italic">M. Liza</Emphasis> (Pisces,Mugiliformes), Characterized by Significant Karyotype Diversity

Two congeneric mullet species, Mugil liza and M. curema, respectively with an all-uniarmed and an all-biarmed karyotype, were cytogenetically studied by base-specific fluorochrome staining and FISH-mapping of 45S and 5S ribosomal RNA genes (rDNA) and the (TTAGGG)_n telomeric repeats. Whereas 45S rDNA sites might be homeologus in the two species, 5S rDNA sites are not, as they are localized on chromosome arms of different size. In both species, the (TTAGGG)_n telomeric probe hybridized to natural telomeres and was found scattered along the NORs. In metacentric chromosomes of M. curema, no pericentromeric signals of the telomeric probe were detected. Data are discussed in relation to the karyotype evolution in Mugilidae and to the mechanisms and the evolutionary implications of Robertsonian rearrangements in M. curema.     

Distinct domains in the CArG-box binding factor A destabilize tetraplex forms of the fragile X expanded sequence d(CGG)n

Formation of hairpin or tetraplex structures of the FMR1 gene d(CGG)_n sequence triggers its expansion, setting off fragile X syndrome. In searching for proteins that destabilize d(CGG)_n secondary structures we purified from rat liver quadruplex telomeric DNA binding protein 42 (qTBP42) that disrupts G′2 bimolecular tetraplex d(CGG)_n while paradoxically stabilizing the G′2 structure of the telomeric sequence d(TTAGGG)_n. Based on peptide sequence homology of qTBP42 and mouse CArG-box binding factor A (CBF-A), we provide direct evidence that recombinant CBF-A protein is physically and immunochemically indistinguishable from qTBP42 and that it too destabilizes G′2 d(CGG)_n while stabilizing G′2 d(TTAGGG)_n. We inquired whether CBF-A employs the same or different domains to differentially interact with G′2 d(CGG)_n and G′2 d(TTAGGG)_n. Mutant CBF-A proteins that lack each or combinations of its five conserved motifs: RNP1₁, RNP1₂, RNP2₁, RNP2₂ and ATP/GTP-binding box were tested for their G′2 d(CGG)_n destabilization and G′2 d(TTAGGG)_n stabilization activities. We find that either RNP1₁ or the ATP/GTP motifs are necessary and sufficient for G′2 d(CGG)_n destabilization whereas RNP2₁ suppresses destabilization by either one of these two motifs. Neither RNP1₁ nor the ATP/GTP motif are required for G′2 d(TTAGGG)_n stabilization. Hence, CBF-A employs different domains to destabilize G′2 d(CGG)_n or stabilize G′2 d(TTAGGG)_n.     

Intrachromosomal location of the telomeric repeat (TTAGGG)n

Eukaryotic telomeres are specialized DNA-protein structures that are thought to ensure chromosomal stability and complete replication of the chromosome ends. All telomeres which have been studied consist of a tandem array of G-rich repeats which seem to be sufficient for telomere function. Originally, the human telomeric repeat (TTAGGG)_n was assumed to be exclusively located at the very end of all human chromosomes. More recent evidence, however, suggests an extension into proterminal regions. Very little is known about the interstitial distribution of telomeric repeats. Here we present evidence for the presence of (TTAGGG)_n repeats in internal loci on the long and short arms of different human chromosomes. In addition, we studied the genomic organization of these repeats in more detail and discuss possible functions of interstitial telomeric repeats in the human genome.     

Identification of high affinity Tbf1p-binding sites within the budding yeast genome

The yeast TBF1 gene is essential for mitotic growth and encodes a protein that binds the human telomere repeats in vitro, although its cellular function is unknown. The sequence of the DNA-binding domain of Tbf1p is more closely related to that of the human telomeric proteins TRF1 and TRF2 than to any yeast protein sequence, yet the functional homologue of TRF1 and TRF2 is thought to be Rap1p. In this study we show that the Tbf1p DNA-binding domain can target the Gal4 transactivation domain to a (TTAGGG)_n sequence inserted in the yeast genome, supporting the model that Tbf1p binds this sub-telomeric repeat motif in vivo. Immunofluorescence of Tbf1p shows a spotty pattern throughout the interphase nucleus and along synapsed chromosomes in meiosis, suggesting that Tbf1p binds internal chromosomal sites in addition to sub-telomeric regions. PCR-assisted binding site selection was used to define a consensus for high affinity Tbf1p-binding sites. Compilation of 50 selected oligonucleotides identified the consensus TAGGGTTGG. Five potential Tbf1p-binding sites resulting from a search of the total yeast genome were tested directly in gel shift assays and shown to bind Tbf1p efficiently in vitro, thus confirming this as a valid consensus for Tbf1p recognition.     

K+ and Na+-Induced Self-Assembly of Telomeric Oligonucleotide d(TTAGGG)n

Abstracts

The telomeric DNA oligomers, d(TTAGGG)_n where n=1, 2, 4, could self-associate into the multi-stranded structures in appropriate condition and exhibited a different CD spectra. The present of Na⁺ was more advantage to facilitate the formation of anti-parallel conformation, but the present of K⁺ enhanced their thermal stability. Spectroscopic analysis of 3, 3′- diethyloxadicarbocyanine (DODC) showed the formation of hairpin quadruplex structures for d(TTAGGG)₂ and d(TTAGGG)₄, but d(TTAGGG) could not. The four-stranded tetraplexes and branched nanowire formed in the present of K⁺ or Na⁺ alone were observed by atomic force microscopy (AFM). The ability to self-assemble of d(TTAGGG)_n into four-stranded tetraplexes and nanowires depends strongly on the number of repeating units and ionic environment. A model to explain how these structures formed is proposed.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

The solution structures of higher-order human telomere G-quadruplex multimers

Human telomeres contain the repeat DNA sequence 5′-d(TTAGGG), with duplex regions that are several kilobases long terminating in a 3′ single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate models proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5′-d(TTAGGG)_n, with n = 8, 12 and 16, fold into multimeric structures containing the maximal number (2, 3 and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of ∼34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.     

Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline

A number of different processes that impact on telomere length dynamics have been identified but factors that affect the turnover of repeats located proximally within the telomeric DNA are poorly defined. We have identified a particular repeat type (CTAGGG) that is associated with an extraordinarily high mutation rate (20% per gamete) in the male germline. The mutation rate is affected by the length and sequence homogeneity of the (CTAGGG)_n array. This level of instability was not seen with other sequence-variant repeats, including the TCAGGG repeat type that has the same composition. Telomeres carrying a (CTAGGG)_n array are also highly unstable in somatic cells with the mutation process resulting in small gains or losses of repeats that also occasionally result in the deletion of the whole (CTAGGG)_n array. These sequences are prone to quadruplex formation in vitro but adopt a different topology from (TTAGGG)_n (see accompanying article). Interestingly, short (CTAGGG)₂ oligonucleotides induce a DNA damage response (γH2AX foci) as efficiently as (TTAGGG)₂ oligos in normal fibroblast cells, suggesting they recruit POT1 from the telomere. Moreover, in vitro assays show that (CTAGGG)_n repeats bind POT1 more efficiently than (TTAGGG)_n or (TCAGGG)_n. We estimate that 7% of human telomeres contain (CTAGGG)_n repeats and when present, they create additional problems that probably arise during telomere replication.     

Mapping of ribosomal DNA and (TTAGGG)n telomeric sequence by FISH in the bivalve Patinopecten yessoensis (Jay, 1857)

The chromosome set of Patinopecten yessoensis (Jay, 1857) wascharacterized using Giemsa staining, DAPI staining and fluorescencein situ hybridization (FISH) with three repetitive DNA probes[18S–28S rDNA, 5S rDNA and telomeric (TTAGGG)_n]. DAPIstaining showed that AT-rich regions were located on the centromereof almost all chromosomes and interstitial banding was not observed.FISH showed that 18S–28S rDNA spread over the short armsof two subtelocentric chromosome pairs and 5S rDNA was locatedon the long arm of one subtelocentric chromosome pair. SequentialFISH demonstrated that 18S–28S and 5S rDNA were locatedon different chromosomes. FISH also showed that the vertebratetelomeric sequence (TTAGGG)_n was located on both ends of eachchromosome and no interstitial signals were detected. Sequential18S–28S rDNA and (TTAGGG)_n FISH indicated that repeatedunits of the two multicopy families were closely associatedon the same chromosome pair. (Received 4 January 2007; accepted 1 September 2007)     

Heterochromatin and microsatellites detection in karyotypes of four sea turtle species: Interspecific chromosomal differences

The wide variation in size and content of eukaryotic genomes is mainly attributed to the accumulation of repetitive DNA sequences, like microsatellites, which are tandemly repeated DNA sequences. Sea turtles share a diploid number (2n) of 56, however recent molecular cytogenetic data have shown that karyotype conservatism is not a rule in the group. In this study, the heterochromatin distribution and the chromosomal location of microsatellites (CA)_n, (GA)_n, (CAG)_n, (GATA)_n, (GAA)_n, (CGC)_n and (GACA)_n in Chelonia mydas, Caretta caretta, Eretmochelys imbricata and Lepidochelys olivacea were comparatively investigated. The obtained data showed that just the (CA)_n, (GA)_n, (CAG)_n and (GATA)_n microsatellites were located on sea turtle chromosomes, preferentially in heterochromatic regions of the microchromosomes (mc). Variations in the location of heterochromatin and microsatellites sites, especially in some pericentromeric regions of macrochromosomes, corroborate to proposal of centromere repositioning occurrence in Cheloniidae species. Furthermore, the results obtained with the location of microsatellites corroborate with the temperature sex determination mechanism proposal and the absence of heteromorphic sex chromosomes in sea turtles. The findings are useful for understanding part of the karyotypic diversification observed in sea turtles, especially those that explain the diversification of Carettini from Chelonini species.     

Molecular and chromosomal analysis of ribosomal cistrons in two cartilaginous fish, <Emphasis Type="Italic">Taeniura lymma</Emphasis> and <Emphasis Type="Italic">Raja montagui</Emphasis> (Chondrichthyes,Batoidea)

We used silver nitrate staining, CMA₃ and FISH to study the chromosomal localization of both the major ribosomal genes and the nucleolar organizer regions as well as that of the minor ribosomal genes (5S rDNA) in two species of Batoidea, Taeniura lymma (Dasyatidae) and Raja montagui (Rajidae). In both species, all the metaphases examined showed the presence of multiple NOR-bearing sites, while the gene for 5S rRNA proved to be localized on two chromosome pairs. Furthermore, one of the two 5S rDNA sites in T. lymma was shown to be co-localized with the major ribosomal cluster. The presence of multiple nucleolar organizer regions in the two species might be interpreted as being the result of intraspecific polymorphisms, or as a phenomenon of the amplified transposition of mobile elements of the genome. We also determined the nucleotide sequence of the 5S rRNA gene, consisting of 564 bp in R. montagui and 612 bp in T. lymma. We also found TATA-like and (TGC)_n trinucleotides, (CA)_n dinucleotides and (GTGA)_n tetranucleotides, which probably influence gene regulation.     

Ordered arrangement and rearrangement of chromosomes during spermatogenesis in two species of planarians (Plathelminthes)

The pattern of distribution of telomeric DNA (TTAGGG), 28S rDNA, and 5S rDNA has been studied using fluorescence in situ hybridization (FISH) and primed in situ labelling during spermatogenesis and sperm formation in the filiform spermatozoa of two species of planarians, Dendrocoelum lacteum and Polycelis tenuis (Turbellaria, Plathelminthes). In both species, the positions of FISH signals found with each probe sequence are constant from cell to cell in the nuclei of mature sperm. Chromosome regions containing 5S and 28S rDNA genes are gathered in distinct bundles of spiral form. In early spermatids with roundish nuclei, the sites of a given sequence on different chromosomes remain separate. Centromeres (marked by 5S rDNA) gather into a single cluster in the central region of the slightly elongated sperm nucleus. During spermatid maturation, this cluster migrates to the distal pole of the nucleus. In Polycelis, telomeric sites gather into three distinct clusters at both ends and in the middle of the moderately elongated nucleus. These clusters retain their relative positions as the spermatid matures. All the chromosome ends bearing 28S rDNA gather only into the proximal cluster. Our data suggest that structures in the nucleus selectively recognise chromosome regions containing specific DNA sequences, which helps these regions to find their regular places in the mature sperm nucleus and causes clustering of the sites of these sequences located on different chromosomes. This hypothesis is supported by observations on elongated sperm of other animals in which a correlation exists between ordered arrangement of chromosomes in the mature sperm nucleus and clustering of sites of the same sequence from different chromosomes during spermiogenesis. Received: 15 December 1997; in revised form: 24 March 1998 / Accepted: 14 April 1998     

Chromosome analysis and FISH mapping of ribosomal DNA (rDNA), telomeric (TTAGGG)n and (GATA)n repeats in the leech Haemopis sanguisuga (L.) (Annelida: Hirudinea)

In the present paper the chromosome complement (n = 13; 2n = 26) of the common leech Haemopis sanguisuga (L.) (Annelida: Hirudinea: Hirudinidae) was analyzed using banding techniques and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG) _n and (GATA) _n ]. FISH with the rDNA probe consistently mapped major ribosomal clusters (18S–28S rDNA) in the pericentromeric region of one large metacentric chromosome pair; this region, which consisted of heterochromatin rich in GC base pairs, was preferentially stained by silver nitrate (Ag-NOR). The (TTAGGG) _n telomeric probe was hybridized with the termini of nearly all chromosomes, whereas the (GATA) _n probe did not label any chromosome areas.     

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.