An evolutionary change in telomere sequence motif within the plant section Asparagales had significance for telomere nucleoprotein complexes

In association with a phylogenetic tree of Asparagales, our previous results showed that a distinct clade included plant species where the ancestral, Arabidopsis-type of telomeric repeats (TTTAGGG)n had been partially, or fully, replaced by the human-type telomeric sequence (TTAGGG)n. Telomerases of these species synthesize human repeats with a high error rate in vitro. Here we further characterize the structure of telomeres in these plants by analyzing the overall arrangement of major and minor variants of telomeric repeats using fluorescence in situ hybridization on extended DNA strand(s). Whilst the telomeric array is predominantly composed of the human variant of the repeat, the ancestral, Arabidopsis-type of telomeric repeats was ubiquitously observed at one of the ends and/or at intercalary positions of extended telomeric DNAs. Another variant of the repeat typical of Tetrahymena was observed interspersed in about 20% of telomerics. Micrococcal nuclease digestions indicated that Asparagales plants with a human-type of telomere have telomeric DNA organised into nucleosomes. However, unexpectedly, the periodicity of the nucleosomes is not significantly shorter than bulk chromatin as is typical of telomeric chromatin. Using electrophoretic mobility shift assays we detected in Asparagales plants with a human type of telomere a 40-kDa protein that forms complexes with both Arabidopsis- and human-type G-rich telomeric strands. However, the protein shows a higher affinity to the ancestral Arabidopsis-type sequence. Two further proteins were found, a 25-kDa protein that binds specifically to the ancestral sequence and a 15-kDa protein that binds to the human-type telomeric repeat. We discuss how the organisation of the telomere repeats in Asparagales may have arisen and stabilised the new telomere at the point of mutation.     

Minisatellite telomeres occur in the family Alliaceae but are lost in Allium

Although telomere sequences are considered to be highly conserved, there are switch-points in plant telomere evolution that are congruent with species' phylogenies. When Asparagales diverged, the Arabidopsis-type telomeric minisatellite repeat (TTTAGGG)(n) was first replaced by a human-type (TTAGGG)(n) repeat, and both were lost in Allium cepa (Alliaceae). We aimed to discover (1) when this loss occurred during divergence of Alliaceae and, (2) if (TTAGGG)(n) repeats were replaced by other known telomeric minisatellites. Slot-blot hybridization, fluorescent in situ hybridization, BAL31 digestion, asymmetric PCR, and cloning were used to identify and localize candidate telomeric sequences in species of Nothoscordum, Miersia, Ipheion, Tulbaghia, Gethyum, Gilliesia, Leucocoryne, Tristagma, and representatives of the three major Allium clades. Alliaceae genera other than Allium have human (TTAGGG)-type telomeric repeats that form telomeres. In Allium, only Tetrahymena-type (TTGGGG) repeats were ubiquitous in the genome, but they were not localized to telomeres. Likewise, the consensus telomeric repeats in Arabidopsis, human, Bombyx (TTAGG), Chlamydomonas (TTTTAGGG), and Oxytricha (TTTTGGGG) are absent in Allium telomeres. Alliaceae with human-type telomeres share telomere structures with related Asparagales species. We demonstrate that in the Allium ancestor human-type telomeric repeats were lost from telomeres and were not replaced by any investigated alternative minisatellite repeats. However, human and other types of minisatellite telomeric repeats are interspersed in some Allium genomes and their genomic signatures coincide with Allium clades.     

Karyotype analysis in Hyacinthella dalmatica (Hyacinthaceae) reveals vertebrate-type telomere repeats at the chromosome ends.

Chromosome analysis of three different populations of Hyacinthella dalmatica (Lallem.) Trinajsti?, an endemic species of the coastal region of southeastern Europe, showed a unique chromosome number, 2n = 2x = 20, and bimodal karyotype with one large and nine smaller pairs of chromosomes. Staining with fluorochromes CMA3 (chromomycin A3) and DAPI (4,6-diamidino-2-phenylindole) revealed heterochromatic regions associated with NORs, centromeres, and several interstitial heterochromatic bands on the longest chromosome pair. Double-target FISH with two ribosomal DNA probes revealed one locus of 5S rRNA genes in the pericentromeric region of chromosome pair 3 and one locus of 18S-5.8S-26S rRNA genes on the short arm of chromosome pair 4 in all plants and populations analyzed. Southern hybridization analysis and FISH experiments demonstrated that the distal ends of H. dalmatica chromosomes contain the vertebrate telomere (5'-TTAGGG-3') repeat type rather than the Arabidopsis (5'-TTTAGGG-3') heptamer, and so suggest that this Asparagales species along with Aloe and Othocallis contains the vertebrate-type telomere repeat.     

Chromosome termini of the monocot plant Othocallis siberica are maintained by telomerase, which specifically synthesises vertebrate-type telomere sequences

Lack of Arabidopsis-type T3AG3 telomere sequences has recently been reported for the majority of investigated taxa of the monocot order Asparagales. In order to investigate this phenomenon in more detail, we conducted extensive cytogenetic and molecular analyses of the telomeres in Othocallis siberica, a member of this order. Terminal restriction fragment analysis together with Bal31 exonuclease assay showed that chromosome termini in O. siberica are formed by long stretches (more than 10 kbp) of vertebrate-type T2AG3 repeats. In addition, telomerase activity specifically synthesising (T2AG3)n sequence was detected in O. siberica protein extracts by telomerase repeat amplification protocol (TRAP). Fluorescence in situ hybridisation (FISH) revealed the presence of the vertebrate-type T2AG3 telomere sequences at all chromosome termini and at a few additional regions of O. siberica chromosomes, whereas Arabidopsis-type T3AG3 DNA and peptide nucleic acid (PNA) probes did not hybridise to chromosomes of Othocallis, except for polymorphic blocks in chromosomes 2 (interstitial) and 4 (terminal). These interstitial/terminal regions are apparently composed of large blocks of (T2AG3)n and (T3AG3)n DNA and represent a unique example of interspersion of two types of telomeric repeats within one genome. This may be a reflection of the recent evolutionary switch from Arabidopsis- to vertebrate-type telomeric repeats in this plant group.     

Loss and recovery of Arabidopsis-type telomere repeat sequences 5'-(TTTAGGG)(n)-3' in the evolution of a major radiation of flowering plants

Fluorescent in situ hybridization and Southern blotting were used for showing the predominant absence of the Arabidopsis-type telomere repeat sequence (TRS) 5'-(TTTAGGG)(n)-3' (the 'typical' telomere) in a monocot clade which comprises up to 6300 species within Asparagales. Initially, two apparently disparate genera that lacked the typical telomere were identified. Here, we used the new angiosperm phylogenetic classification for predicting in which other related families such telomeres might have been lost. Our data revealed that 16 species in 12 families of Asparagales lacked typical telomeres. Phylogenetically, these were clustered in a derived clade, thereby enabling us to predict that the typical telomere was lost, probably as a single evolutionary event, following the divergence of Doryanthaceae ca. 80--90 million years ago. This result illustrates the predictive value of the new phylogeny, as the pattern of species lacking the typical telomere would be considered randomly placed against many previous angiosperm taxonomies. Possible mechanisms by which chromosome end maintenance could have evolved in this group of plants are discussed. Surprisingly, one genus, Ornithogalum (Hyacinthaceae), which is central to the group of plants that have lost the typical telomere, appears to have regained the sequences. The mechanism(s) by which such recovery may have occurred is unknown, but possibilities include horizontal gene transfer and sequence reamplification.     

Asparagales Telomerases which Synthesize the Human Type of Telomeres

The order of monocotyledonous plants Asparagales is attractive for studies of telomere evolution as it includes three phylogenetically distinct groups with telomeres composed of TTTAGGG (Arabidopsis-type), TTAGGG (human-type) and unknown alternative sequences, respectively. To analyze the molecular causes of these switches in telomere sequence (synthesis), genes coding for the catalytic telomerase subunit (TERT) of representative species in the first two groups have been cloned. Multiple alignments of the sequences, together with other TERT sequences in databases, suggested candidate amino acid substitutions grouped in the Asparagales TERT synthesizing the human-type repeat that could have contributed to the changed telomere sequence. Among these, mutations in the C motif are of special interest due to its functional importance in TERT. Furthermore, two different modes of initial elongation of the substrate primer were observed in Asparagales telomerases producing human-like repeats, which could be attributed to interactions between the telomerase RNA subunit (TR) and the substrate. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Characterization of nucleoprotein complexes in plants with human-type telomere motifs.

A conserved feature of telomeres is the 3'-overhang of their G-rich strand. These G-overhangs function as substrates for telomerase-mediated strand extension, and are critical for end-protection of telomeres. These functions and their regulations are mediated by specific G-overhang binding proteins. In species of the plant order Asparagales, telomere motifs have diverged from a type typical of the plant Arabidopsis thaliana (TTTAGGG)(n) to a type typical of human (TTAGGG)(n). Presumably, this change in motif had an impact on the structure of the telomere and/or the binding of telomeric proteins, including the G-overhang binding proteins. Therefore, we analyse here nucleoprotein complexes formed by protein extracts from plants possessing human-type telomeres (Muscari armeniacum and Scilla peruviana). Proteins were characterized that bind to the G-rich strand of both telomere motifs, or to the ancestral Arabidopsis-type motif alone, but none bound to double-stranded or C-rich complementary strand telomere motifs. We demonstrate the size, sequence-specificity and thermostability of these DNA-binding proteins. We also analysed the formation of complexes from renatured protein fractions after SDS-PAGE (sodium-dodecyl-sulphate polyacrylamide-gel-electrophoresis). We discuss the evolutionary consequences of protein binding flexibility, to act on both ancestral and present telomeric sequences. Of particular interest is that the ancestral repeat, which is thought not to form the telomere, binds the proteins most strongly. These data are discussed in line with other known plant telomere-binding proteins and with the complex nature of the telomere in Asparagales carrying a human-type motif.     

Telomere variability in the monocotyledonous plant order Asparagales

A group of monocotyledonous plants within the order Asparagales, forming a distinct clade in phylogenetic analyses, was reported previously to lack the 'typical' Arabidopsis-type telomere (TTTAGGG)(n). This stimulated us to determine what has replaced these sequences. Using slot-blot and fluorescent in situ hybridization (FISH) to species within this clade, our results indicate the following. 1. The typical Arabidopsis-type telomeric sequence has been partly or fully replaced by the human-type telomeric sequence (TTAGGG)(n). Species in Allium lack the human-type variant. 2. In most cases the human variant occurs along with a lower abundance of two or more variants of the minisatellite sequences (of seven types evaluated), usually these being the consensus telomeric sequence of Arabidopsis, Bombyx (TTAGG)(n) and Tetrahymena (TTGGGG)(n). FISH shows that the variants can occur mixed together at the telomere. 3. Telomerases generate products with a 6 base pair periodicity and when sequenced they reveal predominantly a reiterated human-type motif. These motifs probably form the 'true telomere' but the error rate of motif synthesis is higher compared with 'typical' plant telomerases. The data indicate that the Asparagales clade is unified by a mutation resulting in a switch from synthesis of Arabidopsis-like telomeres to a low-fidelity synthesis of human-like telomeres.     

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.     

Phylogenetic Inferences and the Evolution of Plastid DNA in Campynemataceae and the Mycoheterotrophic <Emphasis Type="Italic">Corsia dispar</Emphasis> D.L Jones &amp; B. Gray (Corsiaceae)

The phylogenetic positions of the families Campynemataceae and Corsiaceae within the order Liliales remains unclear. To date, molecular data from the plastid genome of Corsiaceae has been obtained exclusively from Arachnitis, for which alignment and phylogenetic inference has proved difficult. The extent of gene conservation among mycoheterotrophic species within Corsiaceae remains unknown. To clarify the phylogenetic position of Campynemataceae and Corsiaceae within Liliales, functional plastid-coding genes of species representing both families have been analyzed. Examination of two phylogenetic data sets of plastid genes employing parsimony, maximum-likelihood, and Bayesian inference methods strongly supported both families forming a basal clade to the remaining taxa of Liliales. The first data set consists of five functional plastid-encoded genes (matK, rps7, rps2, rps19, and rpl2) sequenced from Corsia dispar (Corsiaceae). The data set included 31 species representing all families within Liliales, as well as selected orders that are related closely to Liliales (10 outgroup species from Asparagales, Dioscoreales, and Pandanales). The second phylogenetic analysis was based on 75 plastid genes. This data set included 18 species from Liliales, representing major clades within the order, and 10 outgroup species from Asparagales, Dioscoreales, and Pandanales. In this latter data set, Campynemataceae was represented by 60 plastid-encoded genes sequenced from herbarium material of Campynema lineare. A large proportion of the plastid genome of C. dispar was also sequenced and compared to the plastid genomes of photosynthetic plants within Liliales and mycoheterotrophic plants within Asparagales to explore plastid genome reduction. The plastid genome of C. dispar is in the advanced stages of reduction, which signifies its high dependency on mycorrhizal fungi and is suggestive of a loss in photosynthetic ability. Functional plastid genes found in C. dispar may be applicable to other species in Corsiaceae, which will provide a basis for in-depth molecular analyses of interspecies relationships within the family, once molecular data from other members become available.     

单子叶植物高级分类阶元系统演化: matK、rbcL和18S rDNA序列的证据

基于两个叶绿体基因（matK和rbcL）和一个核糖体基因（18S rDNA）的序列分析,对代表了基部被子植物和单子叶植物主要谱系分支的86科126属151种被子植物（单子叶植物58科86属101种）进行了系统演化关系分析。研究结果表明由胡椒目Piperales、樟目Laurales、木兰目Magnoliales和林仙目Canellales构成的真木兰类复合群是单子叶植物的姐妹群。单子叶植物的单系性在3个序列联合分析中得到98％的强烈自展支持。联合分析鉴定出9个单子叶植物主要谱系（广义泽泻目Alismatales、薯蓣目Dioscorcales、露兜树目Pandanales、天门冬目Asparagalcs、百合目Liliales、棕榈目Arecales、禾本目Poales、姜目Zingiberales、鸭跖草目Commelinales）和6个其他被子植物主要谱系（睡莲目Nymphaeales、真双子叶植物、木兰目、樟目、胡椒目、林仙目）。在单子叶植物内,菖蒲目Acorales（菖蒲属Acorus）是单子叶植物最早分化的一个谱系,广义泽泻目（包括天南星科Araceae和岩菖蒲科Toficldiaccae）紧随其后分化出来,二者依次和其余单子叶植物类群构成姐妹群关系。无叶莲科Petrosaviaceac紧随广义的泽泻目之后分化出来,无叶莲科和剩余的单子叶植物类群形成姐妹群关系,并得到了较高的支持率。继无叶莲科之后分化的类群形成两个大的分支：一支是由露兜树目和薯蓣目构成,二者形成姐妹群关系：另一支是由天门冬目、百合目和鸭跖草类复合群组成,三者之间的关系在单个序列分析和联合分析中不稳定,需要进一步扩大取样范围来确定。在鸭跖草类复合群分支内,鸭跖草目和姜目的姐妹群关系在3个序列联合分析和2个序列联合分析的严格一致树中均得到强烈的自展支持,获得的支持率均是100％。但是,对于棕榈目和禾本目在鸭跖草类中的系统位置以及它们和鸭跖草目-姜目之间的关系,有待进一步解决。值得注意的是,无叶莲科与其他单子叶植物类群（除菖蒲目和泽泻目外）的系统关系在本文中获得较高的自展支持率,薯蓣目和天门冬目的单系性在序列联合分析中都得到了较好的自展支持,而这些在以往的研究中通常支持率较低。鉴于菖蒲科和无叶莲科独特的系统演化位置,本文支持将其分别独立成菖蒲目和无叶莲目Petrosavialcs的分类学界定。     

TCAGG,an alternative telomeric sequence in insects

The TTAGG repeat, the only determined telomerase-dependent sequence in the Insecta, is generally reputed to be the canonical telomeric motif within the class. By studying the distribution of telomeric DNAs in 30 coleopteran beetles using Southern hybridization, BAL 31 DNA end-degradation assay and fluorescence in situ hybridization, we showed that arrays built of a TCAGG repeat substitute for (TTAGG)n sequences in all tested species within the superfamily Tenebrionoidea. We also provided the experimental evidence that (TCAGG)n repeats represent the terminal sequences on all chromosomes of the model species Tribolium castaneum. (TCAGG)n repeats are therefore promoted as the first sequence-motif alternative to TTAGG-type chromosome ends in insects. Detection of species negative for both TTAGG and TCAGG reveals that, although widespread, these motifs are not ubiquitous telomeric sequences within the order Coleoptera. In addition, Timarcha balearica proved to be a species that harbors (TTAGG)n repeats, but not at telomeric positions, thus further increasing the complexity of telomeric DNAs. Our experiments discarded CTAGG, CTGGG, TTGGG, and TTAGGG variants as potential replacements in TTAGG/TCAGG-negative species, indicating that chromosome termini of these beetles comprise other form(s) of telomeric sequences and telomere maintenance mechanisms.     

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.     

Telomeric DNA in chromosomes of five opisthorchid species

The analysis of telomere repeat distribution in chromosomes of five opisthorchid species (Opisthorchis felineus (Rivolta, 1884), Opisthorchis viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), Metorchis bilis (Braun, 1890), Clonorchis sinensis (Cobbold, 1875)) was performed with fluorescent in situ hybridization (FISH) of labeled (TTAGGG)n DNA-probe and PNA telomere probe on mitotic and meiotic chromosomes of these species. It was shown that chromosome telomeres of all studied species contain large clusters of (TTAGGG)n telomeric repeats. Interstitial clusters of the (TTAGGG)n repeats have not been revealed in the chromosomes of any studied species even when FISH of PNA telomere probe on pachytene chromosomes was performed. Furthermore interstitial clusters of the (TTAGGG)n repeats have not been detected in the chromosomes of O. viverrini, one of chromosomes of this species is the result of a fusion of two ancestral opisthorchid chromosomes.     

Validation and development of quantitative flow cytometry-based fluorescence in situ hybridization for intercenter comparison of telomere length measurement

BACKGROUND: Telomeres are highly conserved repeats at the ends of chromosomes that maintain chromosome stability and reflect the replicative potential of cells. Telomere length can be determined by Southern blot hybridization or quantitative fluorescence in situ hybridization (Q-FISH). Recently, two flow cytometry-based (Flow) FISH protocols have been published. METHODS: We compared the telomere length measured by Southern blotting and Flow FISH using standard beads to calibrate and quantify the fluorescence intensity. RESULTS: The telomeric fluorescence of cord blood and peripheral blood mononuclear cells was similar to that reported by other studies. There was a linear relationship between the telomeric fluorescence determined by Flow FISH and the telomere fragment size determined by Southern blotting (r = 0.89; P < 0.001). CONCLUSION: It is important to set up a center-specific curve and select appropriate cell lines for reference. This Q-Flow FISH protocol will facilitate the measurement of telomere length and allow more meaningful comparison of data (in standard fluorescence units or fragment size) between institutes.     

Phylogenetic distribution of TTAGG telomeric repeats in insects.

We examined the presence of TTAGG telomeric repeats in 22 species from 20 insect orders with no or inconclusive information on the telomere composition by single-primer polymerase chain reaction with (TTAGG)6 primers, Southern hybridization of genomic DNAs, and fluorescence in situ hybridization of chromosomes with (TTAGG)n probes. The (TTAGG)n sequence was present in 15 species and absent in 7 species. In a compilation of new and published data, we combined the distribution of (TTAGG)n telomere motif with the insect phylogenetic tree. The pattern of phylogenetic distribution of the TTAGG repeats clearly supported a hypothesis that the sequence was an ancestral motif of insect telomeres but was lost repeatedly during insect evolution. The motif was conserved in the "primitive" apterous insect orders, the Archaeognatha and Zygentoma, in the "lower" Neoptera (Plecoptera, Phasmida, Orthoptera, Blattaria, Mantodea, and Isoptera) with the exception of Dermaptera, and in Paraneoptera (Psocoptera, Thysanoptera, Auchenorrhyncha, and Sternorrhyncha) with the exception of Heteroptera. Surprisingly, the (TTAGG)n motif was not found in the "primitive" pterygotes, the Palaeoptera (Ephemeroptera and Odonata). The Endopterygota were heterogeneous for the occurrence of TTAGG repeats. The motif was conserved in Hymenoptera, Lepidoptera, and Trichoptera but was lost in one clade formed by Diptera, Siphonaptera, and Mecoptera. It was also lost in Raphidioptera, whereas it was present in Megaloptera. In contrast with previous authors, we did not find the motif in Neuroptera. Finally, both TTAGG-positive and TTAGG-negative species were reported in Coleoptera. The repeated losses of TTAGG in different branches of the insect phylogenetic tree and, in particular, in the most successful lineage of insect evolution, the Endopterygota, suggest a backup mechanism in the genome of insects that enabled them frequent evolutionary changes in telomere composition.     

Conservation of (TTAGG)(n) telomeric sequences among ants (Hymenoptera,Formicidae)

To determine the telomere sequence in Tapinoma nigerrimum, we carried out in situ hybridization using TTAGGG and TTAGG repeat polymerase chain reaction (PCR)-generated probes. No hybridization signals were found when TTAGGG was used as a probe. However, strong signals were observed at the end of the chromosomes with the TTAGG probe. Southern blot analysis carried out on genomic DNA using TTAGG as a probe showed a strong hybridization signal even under highly stringent conditions. Similar results were obtained in Southern blot analysis carried out on genomic DNA of 19 species of ants belonging to three different subfamilies. In accordance with all the results shown in this article, the TTAGG repeat seems to be the major component of the telomere sequence in the majority of ant species.     

AT-rich sequences containing Arabidopsis-type telomere sequence and their chromosomal distribution in Pinus densiflora

Japanese red pine Pinus densiflora has 2 n=24 chromosomes and after FISH-detection of Arabidopsis-type (A-type) telomere sequences, many telomere signals were observed on these chromosomes at interstitial and proximal regions in addition to the chromosome ends. These interstitial and proximal signal sites were observed as DAPI-positive bands, suggesting that the interstitial and proximal telomere signal sites are composed of AT-rich highly repetitive sequences. Four DNA clones (PAL810, PAL1114, PAL1539, PAL1742) localized at the interstitial telomere signals were selected from AluI-digested genomic DNA library using colony blot hybridization probed with A-type telomere sequences and characterized using FISH and Southern blot hybridization. The AT-contents of these selected four clones were 60.8–76.3%, and repeat units of the telomere sequence and degenerated telomere sequences were found in their nucleotide sequences. Except for two sites of PAL1114, FISH signals of the four clones co-localized with interstitial and proximal A-type telomere sequence signals. FISH signals a showed similar distribution pattern, but the patterns of signal intensity were different among the four clones. PAL810, PAL1539 and PAL 1742 showed similar FISH signal patterns, and the differences were only with respect to the signal intensity of some signal sites. PAL1114 had unique signals that appeared on chromosomes 7 and 10. Based on results of the Southern blot hybridization these four sequences are not arranged tandemly. Our results suggest that the interstitial A-type telomere sequence signal sites were composed of a mixture of several AT-rich repetitive sequences and that these repetitive sequences contained A-type telomere sequences or degenerated A-type telomere sequence repeats.     

Measurement of telomere length in haematopoietic cells using in situ hybridization techniques

The DNA of human chromosomes terminates in several kilobases of telomere repeats that are gradually lost with; age and with replication in vitro. Defective telomere maintenance has been shown to be causally linked to cell cycle exit and apoptosis. In order to overcome the limitations imposed by Southern blotting, we have established a quantitative fluorescence in situ hybridization (Q-FISH) technique. This technique allows estimation of telomere length in specific chromosome arms from metaphase cell preparations. Furthermore, we have extended quantitative in situ hybridization to flow cytometry (flow FISH) in order to obtain information on the mean telomere repeat content in suspended cells. Telomere length in granulocytes, monocytes, CD8 and CD4 T lymphocytes and natural killer cells was found to differ slightly in the peripheral blood of adults. However, strikingly longer telomeres were observed in B lymphocytes (approximately 1.3 kb longer), suggesting a functional role for telomere maintenance in this cell subset. In summary, Q-FISH and flow FISH represent new methods for measuring telomere length in single cells and allow studies of telomere dynamics in haematopoietic subpopulations at various stages of normal and abnormal antigen responses.     

Diverse patterns of the tandem repeats organization in rye chromosomes

Although the monomer size, nucleotide sequence, abundance and species distribution of tandemly organized DNA families are well characterized, little is known about the internal structure of tandem arrays, including total arrays size and the pattern of monomers distribution. Using our rye specific probes, pSc200 and pSc250, we addressed these issues for telomere associated rye heterochromatin where these families are very abundant. Fluorescence in situ hybridization (FISH) on meiotic chromosomes revealed a specific mosaic arrangement of domains for each chromosome arm where either pSc200 or pSc250 predominates without any obvious tendency in order and size of domains. DNA of rye-wheat monosomic additions studied by pulse field gel electrophoresis produced a unique overall blot hybridization display for each of the rye chromosomes. The FISH signals on DNA fibres showed multiple monomer arrangement patterns of both repetitive families as well as of the Arabidopsis-type telomere repeat. The majority of the arrays consisted of the monomers of both families in different patterns separated by spacers. The primary structure of some spacer sequences revealed scrambled regions of similarity to various known repetitive elements. This level of complexity in the long-range organization of tandem arrays has not been previously reported for any plant species. The various patterns of internal structure of the tandem arrays are likely to have resulted from evolutionary interplay, array homogenization and the generation of heterogeneity mediated by double-strand breaks and associated repair mechanisms.