Telomere-specific non-LTR retrotransposons and telomere maintenance in the silkworm, <Emphasis Type="BoldItalic">Bombyx mori</Emphasis>

Most insects have telomeres that consist of pentanucleotide (TTAGG) telomeric repeats, which are synthesized by telomerase. However, all species in Diptera so far examined and several species in other orders of insect have lost the (TTAGG)_n repeats, suggesting that some of them recruit telomerase-independent telomere maintenance. The silkworm, Bombyx mori, retains the TTAGG motifs in the chromosomal ends but expresses quite a low level of telomerase activity in all stages of various tissues. Just proximal to a 6–8-kb stretch of the TTAGG repeats in B. mori, more than 1000 copies of non-LTR retrotransposons, designated TRAS and SART families, occur among the telomeric repeats and accumulate. TRAS and SART are abundantly transcribed and actively retrotransposed into TTAGG telomeric repeats in a highly sequence-specific manner. They have three possible mechanisms to ensure specific integration into the telomeric repeats. This article focuses on the telomere structure and telomere-specific non-LTR retrotransposons in B. mori and discusses the mechanisms for telomere maintenance in this insect.     

Telomeric and interstitial telomeric sequences in holokinetic chromosomes of Lepidoptera: Telomeric DNA mediates association between postpachytene bivalents in achiasmatic meiosis of females

Telomeres, besides their main role in the protection and maintenance of chromosome ends, have several other vital functions in the cell cycle. We studied their role in the achiasmatic meiosis of female Lepidoptera, insects with holokinetic chromosomes. By fluorescence in-situ hybridization (FISH) with the insect telomeric probe, (TTAGG) _n , we mapped the distribution of telomeric and interstitial telomeric sequences (ITS) in female meiotic chromosomes of two species, Orgyia antiqua with a reduced chromosome number (2n=28) and Ephestia kuehniella mutants, possessing a radiation-induced chromosome fusion in the genome (2n=59). In addition to the strong typical telomeric signals, O. antiqua displayed weaker hybridization signals in interstitial sites of pachytene bivalents. The observed ITS most probably reflect remnants of chromosomal rearrangements and support the hypothesis that the Orgyia karyotype had arisen by multiple fusions of ancestral chromosomes. On the other hand, the absence of ITS in the chromosome fusion of Ephestia indicated the loss of telomeres before the two original chromosomes fused. When the telomeric probe was amplified by enzymatic reaction with tyramid, the number of ITS observed increased in Orgyia, and a few ITS were also observed in several chromosomes of Ephestia but not in the fused chromosome. This suggests that the genomes of both species also contain ITS other than those originating from chromosome fusions. The analysis of female meiotic prophase I revealed non-homologous associations of postpachytene bivalents mediated by telomeric DNA, which were not observed in the pachytene stage. Surprisingly, in early postpachytene nuclei the telomeric associations also involved ITS, whereas later postpachytene nuclei displayed chains of bivalents interconnected only by true telomeres. This finding favours a hypothesis that telomeric associations between bivalents play a role in chromosome segregation in the achiasmatic meiosis of female Lepidoptera. This revised version was published online in July 2006 with corrections to the Cover Date.     

Telomeric (TTAGGG)n Sequences Are Associated With Nucleolus Organizer Regions (NORs) in the Wood Lemming

The distribution of the (TTAGGG)_n telomeric sequence was studied in chromosomes of the wood lemming, Myopus schisticolor, by fluorescence in-situ hybridization. As expected, the hybridization signals were observed at telomeres of all chromosomes. However, quite a number of interstitial telomeric sites were present in the pericentric heterochromatic regions. Consistent strong hybridization signals were also seen at one terminus of chromosomes 5, 7 and 12–15. By post-hybridization G-banding and silver-staining, the large blocks of the telomeric sequences on chromosomes 5 and 12 were localized to nucleolus organizer regions (NORs). This revised version was published online in August 2006 with corrections to the Cover Date.     

The evolutionary origin of insect telomeric repeats, (TTAGG)<Subscript><Emphasis Type="Italic">N</Emphasis></Subscript>

The (TTAGG)n sequence is supposed to be an ancestral DNA motif of telomeres in insects. Here we examined the occurrence of TTAGG telomeric repeats in other arthropods and their close relatives by Southern hybridization of genomic DNAs and fluorescence in-situ hybridization (FISH) of chromosomes with (TTAGG)n probes or, alternatively, with the 'vertebrate' telomeric probe, (TTAGGG)n. Our results show that the (TTAGG)n motif is conserved in entognathous hexapods (Diplura and Collembola), crustaceans (Malacostraca, Branchiura, Pentastomida, and Branchiopoda), myriapods (Diplopoda and Chilopoda), pycnogonids, and most chelicerates (Palpigradi, Amblypygi, Acari, Opiliones, Scorpiones, Pseudoscorpiones, and Solifugae) but not in spiders (Araneae). The presence of TTAGG repeats in these groups suggests that the sequence is an ancestral motif of telomeres not only in insects but in Arthropoda. We failed, however, to detect the TTAGG repeats in close relatives of the arthropods, Tardigrada and Onychophora. But while Onychophora had the 'vertebrate' (TTAGGG)n motif instead, the Tardigrada did not. The (TTAGG)n motif probably evolved from the (TTAGGG)n motif. Based on our and compiled data, we presume that the 'vertebrate' motif (TTAGGG)n is an ancestral motif of telomeres in bilaterian animals and possibly also in the superclade including animals, fungi and amoebozoans.     

Localization of a vertebrate telomeric sequence in the chromosomes of two marine worms (phylum Annelida: class polychaeta)

Using the fluorescencein situ hybridization (FISH) technique, the presence of the vertebrate telomeric sequence (TTAGGG) _n was found in the chromosomes of two marine polychaetes belonging to two separate orders: one errant,Platynereis dumerilii (family Nereidae), and the other sessile,Pomatoceros lamarckii (family Serpulidae). This sequence was exclusively present at the ends of the chromosomes in both species.     

Chromosomal localization of the telomeric (TTAGGG)<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> sequence in four species of Armadillo (Dasypodidae) from Argentina: an approach to explaining karyotype evolution in the Xenarthra

The distribution of the vertebrate telomeric sequence (TTAGGG)_n in four species of armadillos (Dasypodidae, Xenarthra), i.e. Chaetophractus villosus (2n = 60), Chaetophractus vellerosus (2n = 62), Dasypus hybridus (2n = 64) and Zaedyus pichiy (2n = 62) was examined by FISH with a peptide nucleic acid (PNA) probe. Besides the expected telomeric hybridization, interstitial (centromeric) locations of the (TTAGGG)n sequence were observed in one chromosome pair of Chaetophractus vellerosus and Zaedyus pichiy, suggesting chromosome fusion of ancestral chromosomes occurring during the evolution of Dasypodidae. In addition, all the species analysed showed one to four apparently telocentric chromosomes, exhibiting only two telomeric signals. However, the immunodetection study of kinetochore proteins on synaptonemal complex spreads from C. villosus showed that the apparently telocentric chromosomes have a tiny short arm that can be resolved only in the more elongated pachytene bivalents. This finding suggests that none of the species of armadillos possess true telocentric chromosomes. Our present results support a reduction in the diploid number by fusion of acrocentrics with loss of chromosome material as a tendency in Dasypodidae.     

A 169-base pair tandem repeat DNA marker for subtelomeric heterochromatin and chromosomal rearrangements in aphids of the Myzus persicae group

Numerous copies of a 169-base pair DNA sequence (Myzus persicae group repeat; MpR) occur at subtelomeric locations on all chromosomes of three members of the Myzus persicae species group (Myzus persicae, M. antirrhinii, M. certus). MpR occurs in large tandem arrays at both ends of all autosomes of the standard 2n = 12 karyotype, and near one end of the X chromosome (the end opposite to the nucleolar organizer) and is estimated to make up about 5% of the genome (a total of about 200 000 copies). Locations of MpR were compared in various karyotypes to determine the likely nature of the rearrangements (fusions, dissociations, translocations) that are found in this species group which, like other Hemiptera, has holocentric chromosomes that are devoid of morphological markers. Aphid clones heterozygous for autosome dissociations do not have any detectable MpR at 'new' chromosome ends, indicating that this sequence is not involved in 'capping' of chromosomes. However, a clone with a de novo autosome fusion had an interstitial block of MpR marking the point of fusion, and clones heterozygous for an autosomal 1,3 translocation had MpR from autosome 1 translocated to a new site on autosome 3. The isolation from M. antirrhinii of the telomeric repeat TTAGG, which is found in several insect groups, is also reported.     

Meiotic instability of chicken ultra-long telomeres and mapping of a 2.8 megabase array to the W-sex chromosome

The objective of this research was to study the meiotic stability of a subset of chicken telomere arrays, which are the largest reported for any vertebrate species. Inheritance of these ultra-long telomere arrays (200 kb to 3 mb) was studied in a highly homozygous inbred line, UCD 003 (F ≥ 99.9). Analysis of array transmission in four families indicated unexpected heterogeneity and non-Mendelian segregation including high-frequency-generation of novel arrays. Additionally, the largest array detected (2.8 Mb) was female-specific and correlated to the most intense telomeric DNA signal on the W-sex chromosome by fluorescence in situ hybridization (FISH). These results are discussed in regard to the potential functions of the ultra-long telomere arrays in the chicken genome including generation of genetic variation through enhanced recombination, protection against erosion by providing a buffer for gene-dense regions, and sex-chromosome organization.     

Presence of a functional (TTAGG)<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> telomere-telomerase system in aphids

The structure of the telomeres of four aphid species (Acyrthosiphon pisum, Megoura viciae, Myzus persicae and Rhopalosiphum padi) was evaluated by Southern blotting and fluorescent in situ hybridization, revealing that each chromosomal end consists of a (TTAGG) _n repeat. The presence of a telomerase coding gene has been verified successively in the A. pisum genome, revealing that aphid telomerase shares sequence identity ranging from 12% to 18% with invertebrate and vertebrate homologues, and possesses the two main domains involved in telomerase activity. Interestingly, telomerase expression has been verified in different somatic tissues suggesting that, in aphids, telomerase activity is not as restricted as in human cells. The study of telomeres in a M. persicae strain with a variable chromosome number showed that aphid telomerase can initiate the de novo synthesis of telomere sequences at internal breakpoints, resulting in the stabilization of chromosomal fragments.     

Intraspecific variation in the distribution of the interstitial telomeric (TTAGGG)n sequences in Micoureus demerarae (Marsupialia: Didelphidae)

The distribution of the telomeric sequence (TTAGGG)_n was studied in chromosomes of Micoureus demerarae (2n=14), a South American marsupial, by fluorescence in-situ hybridization (FISH). The telomeric repeat sequence was present at both ends of all chromosomes, but also various interstitial telomeric sequences (ITS) were detected in the pericentromeric heterochromatic regions. Intraspecific differences in the number of ITS (2 to 8) were observed without intraindividual variation. The presence of telomere-like sequences in the same regions of constitutive heterochromatin suggest that these segments are not necessarily remnants of true telomeres resulting from chromosome rearrangements but could be part of the satellite DNA.     

Aloe spp. – plants with vertebrate-like telomeric sequences

Chromosome termini of most eukaryotes end in tracks of short tandemly repeated GC-rich sequences, the composition of which varies among different groups of organisms. Plant species predominantly contain (TTTAGGG)_n repeats at their telomeres. However, a few plant species, including members of Alliaceae and Aloe spp. (Asphodelaceae) were found to lack such Arabidopsis-type (T₃AG₃)_n telomeric repeats. Recently, it has been proposed that the lack of T₃AG₃ telomeric repeat sequences extends to all species forming the Asparagales clade. Here, we analysed the composition of Aloe telomeres by single-primer PCR and fluorescence in-situ hybridization (FISH) with directly labelled Arabidopsis-type (TTTAGGG)_28–43 DNA probe, and with vertebrate-type (TTAGGG)_33–50 DNA and a (C₃TA₂)₃ peptide nucleic acid (PNA) probe. It was found that Nicotiana tabacum contained Arabidopsis-type telomeric repeats, while Aloe telomeres lacked the corresponding FISH signals. Surprisingly, FISH with the highly specific vertebrate-type (C₃TA₂)₃ PNA probe resulted in strong T₂AG₃-specific FISH signals at the ends of chromosomes of both Aloe and Nicotiana tabacum, suggesting the presence of T₂AG₃ telomeric repeats in these species. FISH with a long (TTAGGG)_33–50 DNA probe also highlighted Aloe chromosome ends, while this probe failed to reveal FISH signals on tobacco chromosomes. These results indicate the presence of vertebrate-like telomeric sequences at the telomeres of Aloe spp. chromosomes. However, single-primer PCR with (T₂AG₃)₅ primers failed to amplify such sequences in Aloe, which could indicate a low copy number of T₂AG₃ repeats at the chromosome ends and/or their co-orientation and interspersion with other repeat types. Our results suggest that telomeres of plant species, which were thought to lack GC-rich repeats, may in fact contain variant repeat types. This revised version was published online in July 2006 with corrections to the Cover Date.     

Colocalization of (TTAGGG)n telomeric sequences and ribosomal genes in Atlantic eels

The distribution of (TTAGGG)_n telomeric repeats was studied in chromosomes of two Atlantic eels,Anguilla anguilla andA. rostrata. We found that these sequences hybridize to all the telomeres but also to the entire nucleolar organizer region (NOR) localized in both species at the short arm of chromosome 8. This was considered to be due to the interspersion of telomeric sequences within the NOR ones. Whatever the significance of this interspersion may be, it seems to be limited toA. anguilla andA. rostrata since inMuraena helena (family muraenidae), which also belongs to the Anguilliformes, no telomeric hybridization signals were found along the NOR regions.     

Cytogenetic analyses of chromosomal rearrangements in Mus minutoides/musculoides from North-West Zambia through mapping of the telomeric sequence (TTAGGG)n and banding techniques

Three specimens of M. minutoides/musculoides from Zambia were cytogenetically studied through G- and C-banding, DAPI staining and fluorescence in-situ hybridization (FISH) with a (TTAGGG)_n telomeric sequence. Biarmed chromosomes were identified according to the current nomenclature as follows: Rb(2.7), Rb(3.12), Rb(4.5), Rb(6.8), Rb(9.16), and the sex chromosomes Rb(1.X), Rb(1.Y) and Rb(1.X_d), originated from the deleted X chromosome. One female showed the diploid number 2n=24; in the two other individuals, the Rb(9.16) occurred in a heteromorphic condition, and, accordingly, the diploid number was 2n=25. FISH showed the sites of telomeric sequences at telomeres of all the chromosomes, and in an interstitial position at the centromeres of all Robertsonian metacentrics, except one – the Rb(6.8), though the patterns of hybridization varied between chromosomes. Sex chromosome pairs, in the male and females, showed a similar C-banding pattern, but revealed clear differences after FISH. Traces of telomeric sequences were found dispersed in the whole-heterochromatic arm of the Rb(1.X_d). No visible bond between C-positive heterochromatin and telomeric sequences were detected in the other either bi- or uniarmed chromosomes, indicating that they may actually represent retained telomeres in the Robertsonian metacentrics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection of alien chromatin conferring resistance to the beet cyst nematode (Heterodera schachtii Schm.) in cultivated beet (Beta vulgaris L.) using in situ hybridization

Chromatin originating from wild beets of the genus Beta, section Procumbentes, has been investigated in nematode-resistant hybrid-derived lines of sugar beet (Beta vulgaris L.) by in situ hybridization using satellite, telomeric and ribosomal DNA repeats, a yeast artificial chromosome (YAC) and total genomic DNA as probes. The alien chromosome was detected in three monosomic addition lines(2n=18+1) by genomic in situ hybridization. Fluorescence in situ hybridization with a genome-specific satellite repeat and YAC DNA enabled the visualization of Procumbentes chromosomes, and in double-target hybridization it was shown that they do not carry 18S–5.8S–25S rRNA and 5S rRNA genes. The wild beet-specific satellite repeat and the telomere sequence from Arabidopsis thaliana were used to perform a structural analysis of the wild beet chromosome fragments of two resistant fragment addition lines. It was shown that one physical end of the chromosome fragments consists of telomeric repeats. Comparison of fragment sizes indicated that the small chromosome fragments harbouring the resistance gene most likely resulted from the loss of one wild beet chromosome arm and an internal deletion of the remaining arm.This revised version was published online in November 2005 with corrections to the Cover Date.     

Measurement of telomere length on tissue sections using quantitative fluorescence in situ hybridization (Q-FISH)

Loss of telomere repeat sequences occurs after each cell division and telomere shortening has been implicated in cellular senescence. The measurement of telomere length might therefore assess the lifespan of a cell. The aim of this study was to set up and validate a technique enabling the assessment of telomere length on tissue sections. Quantitative fluorescence in situ hybridization (Q-FISH) with telomeric probes was performed on smears and sections from cell preparations or human tissues. The mean fluorescence intensity of telomere spots (FI/spot) was automatically quantified by image analysis. Telomeric restriction fragment (TRF) length was assessed by Southern blotting. There was a positive significant correlation between telomere length, as assessed by Q-FISH, and TRF length determined by Southern blotting in corresponding samples (p < 0.01, r = 0.6 for tissue and p < 0.01, r = 0.79 for cells). FI/spot was higher on smears than on sections, but pairwise comparison showed a significant correlation both for cells and for tissues (r = 0.77, p < 0.001 for cells and p < or = 0.01, r = 0.64 for tissue). Finally, since telomere length is expected to shorten with age, FI/spot was assessed in liver samples according to the age of patients: a negative correlation was demonstrated (r = 0.76, p < 0.01). Inter-assay variation was 7% for Q-FISH performed on tissue sections and 12% on touch preparations. This study shows that Q-FISH can be performed with confidence on fixed frozen tissue sections in order to assess telomere length. It is an easy, accurate, and reproducible in situ method for assessing telomeres in the context of cell type and tissue architecture.     

Heterochromatin Discrimination in Aegilops Speltoides by Simultaneous Genomic In Situ Hybridization

Simultaneous genomic in situ hybridization with probe preannealing (SP-GISH) was used for discriminating Aegilops speltoides chromosome regions by their relatedness to DNA of other species. We used a hybridization mixture of two differently labelled DNAs, one from the species used for chromosome spread preparations and a second from species of different and varying affinity, thus creating a two-colour system showing chromosome regions where alien DNA hybridized. Genomic DNA from A. speltoides was labelled with biotin and preannealed with digoxigenin-labelled total genomic DNA from different accessions of Ae. speltoides, Ae. bicornis, Ae. tauschii and Hordeum spontaneum. The probe mixture was hybridized to mitotic chromosmes of Ae. speltoides. Chromosome regions of preferential hybridization of self-DNA were visualized as green, whereas regions of combined hybridization showed orange–yellow fluorescence. We observed GISH banding patterns with a different degree of green fluorescence along Ae. speltoides chromosomes that directly correlated with evolutionary distance. Small green bands were observed in subtelomeric and telomeric heterochromatic regions using DNA of a different accession of Ae. speltoides, whereas when using DNA of H. spontaneum most regions of the chromosomes, except pericentromeric regions, showed mainly green fluorescence. The resolution and application of the approach to the study of heterochromatin differentiation are discussed.     

Studies on the action of mitomycin C and bleomycin on telomere lengths of human lymphocyte chromosomes

In order to address the problem of the action of cytostatics on chromosome ends, telomere length was measured in human lymphocyte cultures exposed to mitomycin C (MMC) and bleomycin (BLM). Telomere-specific PNA probes were used for the quantitative estimation of the relative telomere length of each individual chromosome by fluorescence in situ hybridization. A high inter-cellular and inter-individual variability of relative telomere lengths was found throughout all experiments. Different responses could be observed with respect to the action of the examined mutagens: The total average fluorescence intensity of labeled telomere repeats was decreased under the action of MMC in two of the experiments, while two revealed no significant alteration. BLM caused no significant change of total average telomeric signal intensity in four, a clear decrease in one of the six experiments, and an increase in another. Although all chromosome ends contributed to the observed trends, single telomeres were affected in a very distinct way. The highest concentration of MMC (1 microg/ml) induced significant shortening of telomeres of the chromosome arms; 2q, 3p, 5q, 7p, 10q, 11p, 13q, 17p, 18p&q, and 21q in two independent experiments. In one BLM experiment with 8 microg/ml, the most distinct decrease (p< or =0.005) of telomeric fluorescence was found at the ends of chromosome arms; 1q, 6p, 17p, 20p&q, and 22q. The increase of telomeric signal intensity affected the telomeres of some individual chromosome arms more than others, e.g. 4q, 6p, 7p, 8p, 13p, and 18q. Although the telomere length of the individual chromosome arms varied widely, clear trends could be observed with respect to the rank which was occupied by telomeric length of the various chromosome arms. The telomeres of the 1p, 3p, 4q, 5p, 12q, and 13q chromosome arms throughout all experiments were among the longest; and those of 13p, 15p, 21p, and 22p were among the shortest telomeres of the karyotype. From these data, it can be concluded that MMC affects the telomeric repeat area of chromosomes more than BLM, which mostly had no significant effect on telomere length in the performed experiments.     

Mosaic telomeric (2;14) association in a child with motor delay

In a 6-year-old girl referred because of mild motor delay and hyperextensible joints, chromosome analysis disclosed a derivative chromosome consisting of end-to-end fusion of chromosomes 2 and 14. Two cell lines existed in which this telomere association was present, one with a 45,XX,tas(2;14)(q37;p11) karyotype and one with a 45,XX,tas(2;14) (q37;q32) karyotype. The cell line with the telomeric fusion of 2q and 14p was present in 90% of the cells; a telomeric fusion of 2q and 14q was seen in the remaining 10% of the cells. In both association complexes, only the centromere of chromosome 14 was active. Fluorescence in situ hybridization with telomere and subtelomere probes disclosed no deletion of chromosomal material. Microsatellite analysis showed that the patient had a normal biparental contribution of chromosomes 14.