Genetic mapping of tandemly repeated telomeric DNA sequences in tomato (Lycopersicon esculentum).

A telomere-associated tandemly repeated DNA sequence of tomato, TGR I, has been used to map telomeres on the tomato RFLP linkage map. Mapping was performed by monitoring the segregation of entire arrays of TGR I from a segregating F2 population using pulsed-field gel electrophoresis (PFGE). With this strategy, four telomeres have been mapped to the ends of the short arm of chromosomes 9 and 12 and the long arms of chromosomes 5 and 11, using a saturated RFLP map of tomato containing approximately 1000 RFLP markers. In all four cases, the TGR I locus maps to the end of the chromosome, and the distance between the most distal single-copy RFLP marker and the telomeric TGR I locus was between 1.6 and 9.6 cM. This indicates that the region close to the telomeres does not show an excessive rate of recombination compared to other regions of the genome and that the RFLP map of tomato is essentially complete and covers the entire genome for all practical purposes. Additionally, the mapping technique presented here should be generally applicable to the mapping of other tandemly repeated DNA sequences.     

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.     

Genetic and physical mapping of barley telomeres

Barley (Hordeum vulgare L.) telomeres were investigated by means of pulsed field gel electrophoresis (PFGE) and in situ hybridization. In situ hybridization showed that a tandemly repeated satellite sequence has a subtelomeric location, and is present at thirteen of the fourteen chromosome ends. PFGE revealed that this satellite sequence is physically close to the telomeric repeat. Pulsed field gel electrophoresis was then used for segregation analysis and linkage mapping of several telomeric and satellite loci in a segregating doubled-haploid population. The telomeric repeat displayed a hypervariable segregation pattern with new alleles occurring in the progeny. Eight satellite and telomeric sites were mapped on an restriction fragment length polymorphism (RFLP)-map of barley, defining the ends of chromosome arms 1L, 2S, 3L, 4S, 4L, 5S and 6. One satellite locus mapped to an interstitial site on the long arm of chromosome 3. The pyhsical location of this locus was confirmed by in situ hybridization to wheat/barley addition line 3.     

Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum

We have examined the structure and chromatin organization of telomeres in Nicotiana tabacum. In tobacco the blocks of simple telomeric repeats (TTTAGGG)_n are many times larger than in other plants, e.g., Arabidopsis thatiana or tomato. They are resolved as multiple fragments 60–160 kb in size (in most cases 90–130 kb) on pulsed-field gel electrophoresis (PFGE) of restriction endonuclease-digested DNA. The major subtelomeric repeat of the HRS60 family forms large homogeneous blocks of a basic 180 by motif having comparable lengths. Micrococcal nuclease (MNase) cleaves tobacco telomeric chromatin into subunits with a short repeat length of 157±5 bp; the subtelomeric heterochromatin characterized by tandemly repeated sequences of the HRS60 family is cut by MNase with a 180 by periodicity. The monomeric and dimeric particles of telomeric and subtelomeric chromatin differ in sensitivity to MNase treatment: the telomeric particles are readily digested, producing ladders with a periodicity of 7 bp, while the subtelomeric particles appear to be rather resistant to intranucleosomal cleavage. The results presented show apparent similarities in the organization of telomeric chromatin in higher plants and mammals.     

Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum

We have examined the structure and chromatin organization of telomeres in Nicotiana tabacum. In tobacco the blocks of simple telomeric repeats (TTTAGGG)_n are many times larger than in other plants, e.g., Arabidopsis thatiana or tomato. They are resolved as multiple fragments 60–160 kb in size (in most cases 90–130 kb) on pulsed-field gel electrophoresis (PFGE) of restriction endonuclease-digested DNA. The major subtelomeric repeat of the HRS60 family forms large homogeneous blocks of a basic 180 by motif having comparable lengths. Micrococcal nuclease (MNase) cleaves tobacco telomeric chromatin into subunits with a short repeat length of 157±5 bp; the subtelomeric heterochromatin characterized by tandemly repeated sequences of the HRS60 family is cut by MNase with a 180 by periodicity. The monomeric and dimeric particles of telomeric and subtelomeric chromatin differ in sensitivity to MNase treatment: the telomeric particles are readily digested, producing ladders with a periodicity of 7 bp, while the subtelomeric particles appear to be rather resistant to intranucleosomal cleavage. The results presented show apparent similarities in the organization of telomeric chromatin in higher plants and mammals.     

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.     

Chromosome-specific alpha satellite DNA from the centromere of human chromosome 16

To examine the molecular organization of DNA sequences located in the centromeric region of human chromosome 16 we have isolated and characterized a chromosome 16-specific member of the alpha satellite DNA family. The probe obtained is specific for the centromere of chromosome 16 by somatic cell hybrid analysis and by fluorescence in situ hybridization and allows detection of specific hybridizing domains in interphase nuclei. Nucleotide sequence analysis indicates that this class of chromosome 16 alpha satellite (D16Z2) is organized as a series of diverged 340-bp dimers arranged in a tandem array of 1.7-kb higher-order repeat units. As measured by pulsed-field gel electrophoresis, the total D16Z2 array spans approximately 1,400-2,000 kb of centromeric DNA. These sequences are highly polymorphic, both by conventional agarose-gel electrophoresis and by pulsed-field gel electrophoresis. Investigation of this family of alpha satellite should facilitate the further genomic, cytogenetic, and genetic analysis of chromosome 16.     

Genetic and physical mapping of telomeres and macrosatellites of rice

Telomeres and telomere-associated satellites of rice were genetically and physically analyzed by pulsed-field gel electrophoresis (PFGE) using Arabidopsis telomeric DNA and rice satellite sequences as probes. We demonstrate that Arabidopsis telomeric sequences hybridize to rice telomeres under the conditions of high stringency. Using the Arabidopsis probe, multiple, discrete telomeric fragments could be identified on pulsed-field gel blots of rice DNAs digested with rare-cutting restriction enzymes. Most of the telomeric bands larger than 300 kb are physically linked with satellite bands as revealed by PFGE. Some of the telomeric and satellite bands segregate in a Mendelian fashion and are highly reproducible. Three such telomeric bands have been mapped to the distal ends of RFLP linkage groups: Telsm-1 on chromosome 8, Telsa-1 on chromosome 9 and Telsm-3 on chromosome 11. One segregating satellite band was mapped to an internal region of chromosome 10. Telomeric fragments were shown not only to be genetically linked to but also physically linked (based on PFGE) to the terminal RFLP markers. The physical distance from telomeric sequences to a distal RFLP marker, r45s gene, on chromosome 9, is 200 kb while the distance from telomeric sequences to RG98, a terminal RFLP marker on chromosome 11, is 260 kb. Physical maps of the telomere regions of chromosome 9 and chromosome 11 are presented.     

Telomere Variation in Xenopus laevis

Eukaryotic telomeres are variable at several levels, from the length of the simple sequence telomeric repeat tract in different cell types to the presence or number of telomere-adjacent DNA sequence elements in different strains or individuals. We have investigated the sequence organization of Xenopus laevis telomeres by use of the vertebrate telomeric repeat (TTAGGG)_n and blot hybridization analysis. The (TTAGGG)_n-hybridizing fragments, which ranged from less than 10 to over 50 kb with frequently cutting enzymes, defined a pattern that was polymorphic between individuals. BAL 31 exonuclease treatment confirmed that these fragments were telomeric. The polymorphic fragments analyzed did not hybridize to 5S RNA sequences, which are telomeric according to in situ hybridization. When telomeric fragments from offspring (whole embryos) were compared to those from the spleens of the parents, the inheritance pattern of some bands was found to be unusual. Furthermore, in one cross, the telomeres of the embryo were shorter than the telomeres of the parents’ spleen, and in another, the male’s testis telomeres were shorter than those of the male’s spleen. Our data are consistent with a model for chromosome behavior that involves a significant amount of DNA rearrangement at telomeres and suggest that length regulation of Xenopus telomeres is different from that observed for Mus spretus and human telomeres.     

Origin and molecular structure of a midget chromosome in a common wheat carrying rye cytoplasm

The origin and molecular structure of the midget chromosome that is retained in a common wheat with rye cytoplasm, were studied by using fluorescent in situ hybridization (FISH). FISH with biotinylated rye genomic DNA as a probe clearly showed that the midget chromosome had originated from certain part(s) of rye chromosome(s). The midget chromosome did not possess sequences similar to wheat rDNA nor to a rye telomeric sequence with a 350 bp repeat unit. However, another repetitive sequence (120 bp family) of rye was found to occur at one end of the midget chromosome. The telomeric repeat sequences from Arabidopsis thaliana cross-hybridized to both ends of the midget chromosome as well as to wheat chromosomes. From the results obtained in this and previous studies, it is assumed that the midget chromosome originated from part of a rye chromosome, most likely the centromeric region of chromosome 1R, and that the telomeric sequences were synthesized de novo.by R. Appels     

The large-scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes.

Repetitive DNA sequences in the terminal heterochromatin of rye (Secale cereale) chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandemly repeated, subtelomeric DNA families with complex but contrasting higher order structural organizations. Polymerase chain reaction analysis with a single primer showed a fraction of the repeat units of one family organized in a "head-to-head" orientation. Such structures suggest evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. In situ hybridization and pulse field gel electrophoresis showed the order of the repeats and the heterogeneity in the lengths of individual arrays. After Xbal digestion and pulse field gel electrophoresis, the telomeric and two subtelomeric clones showed strong hybridization signals from 40 to 100 kb, with a maximum at 50 to 60 kb. We suggest that these fragments define a basic higher order structure and DNA loop domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.     

Molecular cytogenetic analysis of DNA sequences with flanking telomeric repeats in Triticum aestivum cv. Begra.

A cloned genomic DNA fragment (pTa241) formerly derived from a DNA fraction obtained from isolated nuclei of embryos of a Polish cultivar of wheat (Triticum aestivum cv. Begra) comprises a tandem repeat of the telomeric array CCCTAAA, and hybridizes in situ exclusively to the telomeres of all chromosome arms of the somatic chromosome complement of wheat. A second cloned fragment (pTa637) derived from the same fraction is 637 bp long, flanked by 28 bp of the same telomeric repeat unit, and hybridizes in situ to the entire lengths of all the chromosomes of the complement. The same pattern of hybridization was observed when the flanking telomeric sequences were removed. A third DNA fragment (pTa1439), derived from unfractionated genomic DNA and flanked with 62 bp of the same telomeric unit, showed the same patterns of distribution. Together with additional evidence from Southern analysis, these observations were interpreted to mean that these sequences are associated with mobile DNA elements and are distributed widely throughout the genome. The chromosomal distribution of the non-telomeric parts of the clones is consistent with the dispersed genomic distribution characteristic of transposons and retroelements.     

Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops

A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.     

Identification of the telomeric sequence of the acellular slime molds Didymium iridis and Physarum polycephalum.

We have determined the telomeric DNA sequence of the acellular slime molds Didymium iridis and Physarum polycephalum. In both organisms the telomeres consist of tandem repeats of the hexamer 5'(TTAGGG)3'. This sequence was determined by cloning and sequencing the telomeric fragment of the linear extrachromosomal ribosomal DNA from Didymium, as well as direct end labeling and sequencing the rDNA from both organisms. Interestingly, this sequence is identical to the telomeric DNA sequence of the flagellated protozoan Trypanosoma brucei, and suggests that despite the diversity of telomeric sequences previously determined in lower eukaryotes, the necessity to create functional telomeres has led to constraints on these sequences.     

The chromosomal DNA of Streptomyces lividans 66 is linear

Two copies of a DNA sequence similar or identical to one end of the linear plasmid SLP2 were found on the Streptomyces lividans chromosome. Restriction mapping showed that these sequences represented free ends. Electrophoretic retardation and glass-binding studies indicated that the telomeres carry covalently bound proteins. Moreover, the chromosome migrated as an 8Mb linear DNA in pulsed-field gel electrophoresis. A similar finding with the chromosomes of six other Streptomyces species suggested that a linear chromosome may be characteristic of the genus. The S. lividans chromosome can be circularized by joining the two ends by artificial targeted recombination or by spontaneous deletions spanning both telomeres. Thus the chromosome appears to be able to exist, in viable bacteria, as a linear or a circular molecule.     

Genome reorganization in Nicotiana asymmetric somatic hybrids analysed by in situ hybridization

In situ hybridization was used to examine genome reorganization in asymmetric somatic hybrids between Nicotiana plumbaginifolia and Nicotiana sylvestris obtained by fusion of gamma-irradiated protoplasts from one of the parents (donor) with non-irradiated protoplasts from the other (recipient). Probing with biotinylated total genomic DNA from either the donor or the recipient species unequivocally identified genetic material from both parents in 31 regenerant plants, each originating from a different nuclear hybrid colony. This method, termed genomic in situ hybridization (GISH), allowed intergenomic translocations containing chromosome segments from both species to be recognized in four regenerants. A probe homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat (5'-TTTAGGG-3')n, identified telomeres on all chromosomes, including 'mini-chromosomes' originating from the irradiated donor genome. Genomic in situ hybridization to plant chromosomes provides a rapid and reliable means of screening for recombinant genotypes in asymmetric somatic hybrids. Used in combination with other DNA probes, it also contributes to a greater understanding of the events responsible for genomic recovery and restabilization following genetic manipulation in vitro.     

Characterization and organization of DNA sequences adjacent to the human telomere associated repeat (TTAGGG)n.

We present a strategy for the cloning of DNA sequences adjacent to the tandemly repeated DNA sequence (TTAGGG)n. Sequence analysis of 14 independently isolated clones revealed the presence of non-repetitive sequences immediately adjacent to or flanked by blocks of the simple repeat (TTAGGG)n. In addition, we provide sequence information on two previously undescribed tandemly repeated sequences, including a 9 bp repeat and a modification of the (TTAGGG)n repeat. Using different mapping approaches six sub-clones, free of the TTAGGG repeat, were assigned to a single human chromosome. Moreover, in situ hybridization mapped one of these subclones, G2 - 1H, definitively to the telomeric band on chromosome 4q. However, Bal 31 insensitivity suggests a location in a more subterminal region. All the (TTAGGG)n-adjacent unique sequences tested are highly conserved among primates but are not present in other mammalian species. Identification and mapping of TTAGGG-adjacent sequences will provide a refined insight into the genomic organization of the (TTAGGG)n repeat. The isolation of chromosome specific TTAGGG-adjacent sequences from subtelomeric regions of all human chromosomes will serve as important end points for the genetic maps and will be useful for the molecular characterization of chromosomal rearrangements involving telomeres.     

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.     

Telomeric localization of the Arabidopsis‐type heptamer repeat, (TTTAGGG)n,at the chromosome ends in Saccharina japonica (Phaeophyta)

Telomeres generally consist of short repeats of minisatellite DNA sequences and are useful in chromosome identification and karyotype analysis. To date, telomeres have not been characterized in the economically important brown seaweed Saccharina japonica, thus its full cytogenetic research and genetic breeding potential has not been realized. Herein, the tentative sequence of telomeres in S. japonica was identified by PCR amplification with primers designed based on the Arabidopsis‐type telomere sequence (TTTAGGG)_n, which was chosen out of three possible telomeric repeat DNA sequences typically present in plants and algae. After PCR optimization and cloning, sequence analysis of the amplified products from S. japonica genomic DNA showed that they were composed of repeat units, (TTTAGGG)_n, in which the repeat number ranged from 15 to 63 (n = 46). This type of repeat sequence was verified by a Southern blot assay with the Arabidopsis‐type telomere sequence as a probe. The digestion of S. japonica genomic DNA with the exonuclease Bal31 illustrated that the target sequence corresponding to the Arabidopsis‐type telomere sequence was susceptible to Bal31 digestion, suggesting that the repeat sequence was likely located at the outermost ends of the kelp chromosomes. Fluorescence in situ hybridizations with the aforementioned probe provided the initial cytogenetic evidence that the hybridization signals were principally localized at both ends of S. japonica chromosomes. This study indicates that the telomeric repeat of the kelp chromosomes is (TTTAGGG)_n which differs from the previously reported (TTAGGG)_n sequence in Ectocarpus siliculosus through genome sequencing, thereby suggesting distinct telomeres in brown seaweeds.     

Isolation of telomeric DNA from the filamentous fungus Podospora anserina and construction of a self-replicating linear plasmid showing high transformation frequency.

It has been previously shown that linear plasmids bearing Tetrahymena telomeric sequences are able to replicate autonomously in the filamentous fungus Podospora anserina (1). However, autonomous replication occurs in only 50-70% of the transformants, suggesting a defect in the recognition of the Tetrahymena telomeric template by the putative P. anserina telomerase so that only a fraction of entering DNA is stabilized into linear extrachromosomal molecules. We have cloned DNA sequences added to the Tetrahymena (T2G4)n ends of the linear plasmid. Nucleotide sequencing showed that these sequences are exclusively composed of T2AG3 repeat units. Hybridization experiments of Bal31 treated DNA showed that T2AG3 repeats are confined within 200 bp in chromosomal P. anserina telomeres. A new plasmid has been constructed so that after linearization, the terminal sequences contain T2AG3 repeats. This linear molecule transforms P. anserina with a high frequency (up to 1.75 x 10(4) transformants/micrograms), autonomous replication occurs in 100% of the transformants and the plasmid copy number is about 2-3 per nucleus. These results underscore the importance of the telomeric repeat nucleotide sequence for efficient recognition as functional telomeric DNA in vivo and provide the first step toward the development of an artificial chromosome cloning system for filamentous fungi.