Structure and variability of human chromosome ends.

Mammalian telomeres are thought to be composed of a tandem array of TTAGGG repeats. To further define the type and arrangement of sequences at the ends of human chromosomes, we developed a direct cloning strategy for telomere-associated DNA. The method involves a telomere enrichment procedure based on the relative lack of restriction endonuclease cutting sites near the ends of human chromosomes. Nineteen (TTAGGG)n-bearing plasmids were isolated, two of which contain additional human sequences proximal to the telomeric repeats. These telomere-flanking sequences detect BAL 31-sensitive loci and thus are located close to chromosome ends. One of the flanking regions is part of a subtelomeric repeat that is present at 10 to 25% of the chromosome ends in the human genome. This sequence is not conserved in rodent DNA and therefore should be a helpful tool for physical characterization of human chromosomes in human-rodent hybrid cell lines; some of the chromosomes that may be analyzed in this manner have been identified, i.e., 7, 16, 17, and 21. The minimal size of the subtelomeric repeat is 4 kilobases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14 kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14 kb long, telomeres in somatic cells are several kilobase pairs shorter and very heterogeneous in length. Additional telomere reduction occurs in primary tumors, indicating that somatic telomeres are unstable and may continuously lose sequences from their termini.     

Change in the telomere length distribution with age in the Japanese population

Telomeres play a role in cellular aging and they may also contribute to the genetic basis of human aging and longevity. A gradual loss of the telomeric repeat sequences has been reported in adult tissue specimens. This study determined the percentage of telomere restriction fragment in various molecular-sized regions in addition to measuring the average telomere length. Mean telomere restriction fragment (TRF) length was determined by Southern blot analysis using a longer telomeric repeat probe with higher sensitivity. A significant decrease in longer telomere fragments and a quick increase in the shortest fragments were observed, especially in male subjects. There was a tendency that the age-adjusted telomere length was longer in females than that observed in males, while males lose the telomeric sequence faster than females. These data indicated that the percentage of longer telomeres fragments decreased, while the shortest fragments increased quickly with age. In addition, the longest telomere fragments decreased and the short fragments increased with a relatively stable frequency with age. There was also a significant difference in the longest telomere fragment percentage between males and female in their 40s and 50s, whereas no difference was observed in the mean TRF length. Interestingly, the changing rate of the longest and the shortest range group of TRF percentage associated with aging seemed quite different between before and after 50-year old with a gender-related contrast. This contrast implies a drastic change around the age of 50 of unknown factors that affect telomere attrition. The first two authors equally contributed to this article.     

Telomeric repeat sequence of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> consists of dodeca-nucleotides

Four telomeres in the chromosomes of Aspergillus oryzae NFRI1599 were cloned and sequenced. The telomeric repeat sequence of A. oryzae consisted of dodeca-nucleotides: TTAGGGTCAACA. The length of the telomeric repeat tract was 114-136 bp, which corresponds to 9-11 repeats of the dodeca-nucleotide sequence. Compared to a chromosome internal control (18S rDNA), the telomeric sequences were found to be sensitive to BAL31 exonuclease digestion, thus proving that the identified telomeric repeat sequences were located at the most terminal tract of the chromosomes. The length of the telomeric repeat tract of A. oryzae is similar to that of Aspergillus nidulans, whose repeat unit is TTAGGG, indicating that the regulatory mechanism of telomere length might be conserved among Aspergillus species.     

Isolation and characterization of a human telomere.

A method is described that allows cloning of human telomeres in S. cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves. The resulting chimeric yeast-human chromosomes propagate as true linear chromosomes, demonstrating that the human telomere structure is capable of functioning in yeast and suggesting that telomere functions are evolutionarily conserved between yeast and human. One cloned human telomere, yHT1, contains 4 kb of human genomic DNA sequence next to the tandemly repeating TTAGGG hexanucleotide. Genomic hybridizations using both cloned DNA and TTAGGG repeats have revealed a common structural organization of human telomeres. This 4 kb of genomic DNA sequence is present in most, but not all, human telomeres, suggesting that the region is not involved in crucial chromosome-specific functions. However, the extent of common features among the human telomeres and possible similarities in organization with yeast telomeres suggest that this region may play a role in general chromosome behavior such as telomere-telomere interactions. Unlike the simple telomeric TTAGGG repeats, our cloned human genomic DNA sequence does not cross-hybridize with rodent DNA. Thus, this clone allows the identifications of the terminal restriction fragments of specific human chromosomes in human-rodent hybrid cells.     

De novo telomere addition to spacer sequences prior to their developmental degradation in Euplotes crassus

During sexual reproduction, Euplotes crassus precisely fragments its micronuclear chromosomes and synthesizes new telomeres onto the resulting DNA ends to generate functional macronuclear minichromosomes. In the micronuclear chromosomes, the macronuclear-destined sequences are typically separated from each other by spacer DNA segments, which are eliminated following chromosome fragmentation. Recently, in vivo chromosome fragmentation intermediates that had not yet undergone telomere addition have been characterized. The ends of both the macronuclear-destined and eliminated spacers were found to consist of six-base, 3′ overhangs. As this terminal structure on the macronuclear-destined sequences serves as the substrate for de novo telomere addition, we sought to determine if the spacer DNAs might also undergo telomere addition prior to their elimination. Using a polymerase chain reaction approach, we found that at least some spacer DNAs undergo de novo telomere addition. In contrast to macronuclear-destined sequences, heterogeneity could be observed in the position of telomeric repeat addition. The observation of spacer DNAs with telomeric repeats makes it unlikely that differential telomere addition is responsible for differentiating between retained and eliminated DNA. The heterogeneity in telomere addition sites for spacer DNA also resembles the situation found for telomeric repeat addition to macronuclear-destined sequences in other ciliate species.     

Human telomeres contain at least three types of G-rich repeat distributed non-randomly.

Using a combination of different oligonucleotides and restriction enzymes we have examined the gross organisation of repeats within the most distal region of human chromosomes. We demonstrate here that human telomeres do not contain a pure uniform 6 base pair repeat unit but that there are at least three types of repeat. These three types of repeat are present at the ends of most or all human chromosomes. The distribution of each type of repeat appears to be non-random. Each human telomere has a similar arrangement of these repeats relative to the ends of the chromosome. This could reflect differences in the functions that they perform, or might result from the mutation and correction processes occurring at human telomeres. The number of repeat units, the repeat types and arrangement differs at mouse telomeres. Analysing the change in length of the telomeric repeat region between an individuals blood and germline DNA reveals that this is due to variable amounts of the TTAGGG repeat and not the other repeat types. This organization of repeat units at human telomeres will only be confirmed upon the isolation and sequencing of full length (10-15 kb), intact human telomeres.     

Telomere looping in P. sativum (common garden pea)

Telomeres vary greatly in size among plants and, in most higher plants, consist of a long array of 5'-TTTAGGG-3'/3'-AAATCCC-5' (TTTAGGG) repeats. Recently, telomeric DNA in human, mouse, oxytricha, and trypanosome chromosomes have been found arranged into loops (t-loops), proposed to sequester the telomere from unwanted repair events and prevent activation of DNA damage checkpoints. We have asked whether t-loops exist in the higher order plant Pisum sativum (garden pea). DNA was isolated from the shoots and root tips of germinating seeds. Analysis of the telomeric restriction fragments showed that DNA hybridizing to a (TTTAGGG)n probe migrated as a smear centering around 25 kb, and direct sequencing verified the repeat to be (TTTAGGG)n. Total DNA in isolated nuclei was photo-cross-linked, and the telomeric restriction fragments were purified by gel filtration. Electron microscopic (EM) analysis revealed DNA molecules arranged as t-loops with a size distribution consistent with that seen by gel electrophoresis. Some molecules had loops as large as 75 kb. These results show that the arrangement of telomeric DNA into loops occurs in higher plants.     

Measurement of telomere DNA content by dot blot analysis

Telomeres play a central role in human cancer, cardiovascular aging and possibly longevity. However, present methods to measure telomere length are fraught with shortcomings that limit their use. Here, we describe a novel method to measure the relative telomere DNA content by dot blot analysis. In each dot, the DNA content is measured by a DNA stain (Dx) and the telomeric DNA content is measured with a telomeric probe (T). The T normalized for Dx (T/Dx) of each dot is a measure of telomere content. The method requires ～20 ng of DNA per assay. Moreover, the T/Dx data are highly correlated linearly with mean telomere lengths derived from Southern blots of the terminal restriction fragments (r > 0.96, P < 0.0001). The method is also simple to use, has a relatively low interassay coefficient of variation (<6%), retains its precision in moderately degraded DNA and can be forged for high throughput analysis. The method might help researchers and clinicians alike in understanding risks for and extent of human diseases.     

Telomere length measurement-caveats and a critical assessment of the available technologies and tools

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects.     

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.     

Telomeres of the linear chromosomes of Lyme disease spirochaetes: nucleotide sequence and possible exchange with linear plasmid telomeres

Bacteria of the spirochaete genus Borrelia have linear chromosomes about 950 kbp in size. We report here that these linear chromosomes have covalently closed hairpin structures at their termini that are similar but not identical to those reported for linear plasmids carried by these organisms. Nucleotide sequence analysis of the chromosomal telomeric regions indicates that unique, apparently functional genes lie within a  few hundred bp of each of the telomeres, and that there is an imperfect 26 bp inverted repeat at the two telomeres. In addition, we characterize a major chromosomal length polymorphism within the right telomeric regions of various Borrelia isolates, and show that sequences similar to those near the right telomere are often found on linear plasmids in B . burgdorferi ( sensu stricto ) isolates from nature. Sequences similar to a number of other regions of the chromosome, including those near the left telomere, were not found on B . burgdorferi plasmids. These observations suggest that there has been historical exchange of genetic information between the linear plasmids and the right end of the linear chromosome.     

Chinese hamster telomeres are comparable in size to mouse telomeres.

We report here the results of a telomere length analysis in four male Chinese hamsters by quantitative fluorescence in situ hybridization (Q-FISH). We were able to measure telomere length of 64 (73%) of 88 Chinese hamster telomeres. We could not measure telomere length in chromosome 10 or in the short arms of chromosomes 5, 6, 7 and 8 because of the overlaps between the interstitial and terminal telomeric signals. Our analysis in the 73% of Chinese hamster telomeres indicate that their average length is approximately 38 kb. Therefore, Chinese hamster telomeres are comparable in length to mouse telomeres, but are much longer than human telomeres. Similar to previous Q-FISH studies on human and mouse chromosomes, our results indicate that individual Chinese hamster chromosomes may have specific telomere lengths, suggesting that chromosome-specific factors may be involved in telomere length regulation.     

Mouse minor satellite DNA genetically maps to the centromere and is physically linked to the proximal telomere.

As an adjunct to attempts to define functionally important sequences at human centromeres, we have undertaken a long-range physical analysis of these regions in the mouse. Mouse centromeres are usually situated very close to the chromosome ends and are closely associated with minor satellite sequences on the basis of cytological observations. Using pulsed-field gel electrophoresis we find that this satellite DNA is arranged as tandem arrays, predominantly uninterrupted by nonsatellite sequences. These arrays can be released largely intact by digestion with a range of enzymes that generally cleave frequently in non-satellite DNA. The restriction fragments carrying these arrays are polymorphic in size between inbred strains and provide direct markers for mouse centromeres. To illustrate the possible use of these polymorphic markers we have mapped a 1.3-Mb PvuII variant in a set of RI strains to the centromere of Chromosome 7. The minor satellite arrays are very close to the centromeric telomere and physical linkage with terminal repeat sequences can readily be detected, placing many minor satellite arrays on terminal restriction fragments smaller than 1 Mb. The apparent lack of any sizable amount of nonsatellite DNA between the minor satellite and the terminal repeat arrays indicates that many mouse chromosomes are truly telocentric.     

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.     

Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres

In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3' of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3' terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.     

Characterization of Arabidopsis thaliana telomeres isolated in yeast.

In an effort to learn more about the genomic organization of chromosomal termini in plants we employed a functional complementation strategy to isolate Arabidopsis thaliana telomeres in the yeast, Saccharomyces cerevisiae. Eight yeast episomes carrying A. thaliana telomeric sequences were obtained. The plant sequences carried on two episomes, YpAtT1 and YpAtT7, were characterized in detail. The telomeric origins of YpAtT1 and YpAtT7 insert DNAs were confirmed by demonstrating that corresponding genomic sequences are preferentially degraded during exonucleolytic digestion. The isolated telomeric restriction fragments contain G-rich repeat arrays characteristic of A. thaliana telomeres, as well as subterminal telomere-associated sequences (TASs). DNA sequence analysis revealed the presence of variant telomeric repeats at the centromere-proximal border of the terminal block of telomere repeats. The TAS flanking the telomeric G-rich repeat in YpAtT7 corresponds to a repetitive element present at other A. thaliana telomeres, while more proximal sequences are unique to one telomere. The YpAtT1 TAS is unique in the Landsberg strain of A. thaliana from which the clone originated; however, the Landsberg TAS cross-hybridizes weakly to a second telomere in the strain Columbia. Restriction analysis with cytosine methylation-sensitive endonucleases indicated that both TASs are highly methylated in the genome.     

Cytological and molecular analysis of centromere misdivision in maize.

B chromosome derivatives suffering from breaks within their centromere were examined cytologically and molecularly. We showed by high resolution FISH that misdivision of the centromere of a univalent chromosome can occur during meiosis. The breaks divide the centromere repeat sequence cluster. A telocentric chromosome formed by misdivision was found to have the addition of telomeric repeats to the broken centromere. A ring chromosome formed after misdivision occurred by fusion of the broken centromere to the telomere. Pulsed-field electrophoresis analyses were performed on the telocentric and ring chromosomes to identify fragments that hybridize to both the telomeric repeat and the B-specific centromeric repeat. We conclude that healing of broken maize centromeres can be achieved through the mechanisms of addition or fusion of telomeric repeat sequences to the broken centromere.     

Telomere instability in a human cancer cell line.

Telomere maintenance is essential in immortal cancer cells to compensate for DNA lost from the ends of chromosomes, to prevent chromosome fusion, and to facilitate chromosome segregation. However, the high rate of fusion of chromosomes near telomeres, termed telomere association, in many cancer cell lines has led to the proposal that some cancer cells may not efficiently perform telomere maintenance. Deficient telomere maintenance could play an important role in cancer because telomere associations and nondisjunction have been demonstrated to be mechanisms for genomic instability. To investigate this possibility, we have analyzed the telomeres of the human squamous cell carcinoma cell line SQ-9G, which has telomere associations in approximately 75% of the cells in the population. The absence of detectable telomeric repeat sequences at the sites of these telomere associations suggests that they result from telomere loss. The analysis of telomere length by quantitative in situ hybridization demonstrated that, compared to the human squamous cell carcinoma cell line SCC-61 which has few telomere associations, SQ-9G has more extensive heterogeneity in telomere length and more telomeres without detectable telomeric repeat sequences. The dynamics of the changes in telomere length also demonstrated a higher rate of fluctuation in telomere length, both on individual telomeres and coordinately on all telomeres. These results demonstrate that telomere maintenance can play a role in the genomic instability seen in cancer cells.     

A novel minisatellite at a cloned hamster telomere

The ends of eukaryotic chromosomes have special properties and roles in chromosome behavior. Selection for telomere function in yeast, using a Chinese hamster hybrid cell line as the source DNA, generated a stable yeast artificial chromosome clone containing 23 kb of DNA adjacent to (TTAGGG)n, the vertebrate telomeric repeat. The common repetitive element d(GT)n appeared to be responsible for most of the other stable clones. Circular derivatives of the TTAGGG-positive clone that could be propagated in E. coli were constructed. These derivatives identify a single pair of hamster telomeres by fluorescence in situ hybridization. The telomeric repeat tract consists of (TTAGGG)n repeats with minor variations, some of which can be cleaved with the restriction enzyme MnlI. Blot hybridization with genomic hamster DNA under stringent conditions confirms that the TTAGGG tracts are cleaved into small fragments due to the presence of this restriction enzyme site, in contrast to mouse telomeres. Additional blocks of (TTAGGG)n repeats are found 4–5 kb internally on the clone. The terminal region of the clone is dominated by a novel A-T rich 78 bp tandemly repeating sequence; the repeat monomer can be subdivided into halves distinguished by more or less adherence to the consensus sequence. The sequence in genomic DNA has the same tandem organization in probably a single primary locus of >20–30 kb and is thus termed a minisatellite.     

Interaction of an Arabidopsis RNA-binding protein with plant single-stranded telomeric DNA modulates telomerase activity

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a single-stranded 3' overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomere-binding proteins. Although proteins that bind single-stranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants. Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomere-binding protein 1), binds specifically the single-stranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplast-localized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.