SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4–7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9–10×10⁶ base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7–8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G+C content than T. parva or the presence of highly reiterated G+C-rich regions.     

The plasticity of human telomeres demonstrated by a hypervariable telomere repeat array that is located on some copies of 16p and 16q.

Human telomeres are composed of tandem arrays of TTAGGG repeats with many variant repeats at the proximal ends. Comparison of the interspersion of variant and TTAGGG repeats between alleles can be used to study telomere instability, but the difficulty in identifying chromosome-specific sequences close to the start of autosomal telomeres has hampered such investigations. A chromosome end, including a telomere and adjacent sequence, that is polymorphic for its presence or absence in unrelated individuals has been identified. The telomere-adjacent DNA shows strong homology (92-99%) to sequences, including two expressed sequence tags, that are usually located in subterminal regions of human chromosomes but not adjacent to telomeres. Since this chromosome end arose, it has relocated at least once. In Caucasians, it forms the telomere of approximately 6% of 16q and 2% of 16p chromosome arms. The mechanism of relocation is unknown but must have involved the telomere-adjacent DNA rather than the telomere itself, as copies on 16p and 16q share the same telomere-adjacent sequence. The interspersion patterns of TTAGGG with TGAGGG, TTGGGG and non-amplifying repeat sequences revealed extensive allelic variation, such that 47 different alleles were observed among the 50 alleles mapped. Closely related alleles differ by small changes in copy number at blocks of adjacent like repeats, as seen at the Xp/Yp pseudoautosomal telomere. Such differences are compatible with a model in which the majority of mutations arise by intra-allelic mechanisms, in individuals hemizygous for a single copy of the chromosome end.     

Organization of telomeric and sub-telomeric regions of chromosomes from the protozoan parasite Trypanosoma cruzi.

We present here a characterization of the telomeric and subtelomeric regions of Trypanosoma cruzi chromosomes, using three types of recombinants: cosmids from a genomic library, clones obtained by a vector–adaptor protocol, and a recombinant fragment cloned by a Bal31 trimming protocol. The last nine nucleotides of the T. cruzi overhang are 5′-GGGTTAGGG-3′, and there are from 9 to 50 copies of the hexameric repeat 5′-TTAGGG-3′, followed by a 189-bp junction sequence common to all recombinants. The subtelomeric region is made of sequences associated with the gp85/sialidase gene family, and/or sequences derived from SIRE, a retrotransposon-like sequence, and also the retrotransposon L1Tc. We discuss the possible implications of this genome organization.     

Drosophila telomeric retrotransposons derived from an ancestral element that was recruited to replace telomerase

Drosophila telomeres do not have arrays of simple telomerase-generated G-rich repeats. Instead, Drosophila maintains its telomeres by occasional transposition of specific non-long terminal repeat (non-LTR) retrotransposons to chromosome ends. The genus Drosophila provides a superb model system for comparative telomere analysis. Here we present an evolutionary study of Drosophila telomeric elements to ascertain the significance of telomeric retrotransposons (TRs) in the maintenance of Drosophila telomeres. PCR and in silico surveys in the sibling species of Drosophila melanogaster and in more distantly related species show that multiple TRs maintain telomeres in Drosophila. In addition to TRs with two open reading frames (ORFs) capable of autonomous transposition, there are deleted telomeric retrotransposons that have lost their ORF2, which we refer to as half telomeric-retrotransposons (HTRs). The phylogenetic relationship among these telomeric elements is congruent with the phylogeny of the species, suggesting that they have been vertically inherited from a common ancestor. Our results suggest that an existing non-LTR retrotransposon was recruited to perform the cellular function of telomere maintenance.     

Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication

Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired 3'-leading-strand overhangs at the telomeres of replication intermediates. Filling in of these overhangs leaves a terminal protein attached covalently to the 5' DNA ends of mature replicons. We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein, Tap) in this process. Biochemical studies, yeast two-hybrid analysis, and immunoprecipitation/immunodepletion experiments indicate that Tap binds tightly to specific sequences in 3' overhangs and also interacts with Tpg, bringing Tpg to telomere termini. Using DNA microarrays to analyze the chromosomes of tap mutant bacteria, we demonstrate that survivors of Tap ablation undergo telomere deletion, chromosome circularization, and amplification of subtelomeric DNA. Microarray-based chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions, identified amplified chromosomal ORFs adjacent to these endpoints, and quantified the copy number of these ORFs. Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends. Our results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates. They also identify hotspots for the telomeric deletions and subtelomeric DNA amplifications that accompany chromosome circularization.     

The nature of telomere fusion and a definition of the critical telomere length in human cells

The loss of telomere function can result in telomeric fusion events that lead to the types of genomic rearrangements, such as nonreciprocal translocations, that typify early-stage carcinogenesis. By using single-molecule approaches to characterize fusion events, we provide a functional definition of fusogenic telomeres in human cells. We show that approximately half of the fusion events contained no canonical telomere repeats at the fusion point; of those that did, the longest was 12.8 repeats. Furthermore, in addition to end-replication losses, human telomeres are subjected to large-scale deletion events that occur in the presence or absence of telomerase. Here we show that these telomeres are fusogenic, and thus despite the majority of telomeres being maintained at a stable length in normal human cells, a subset of stochastically shortened telomeres can potentially cause chromosomal instability. Telomere fusion was accompanied by the deletion of one or both telomeres extending several kilobases into the telomere-adjacent DNA, and microhomology was observed at the fusion points. This contrasted with telomere fusion that was observed following the experimental disruption of TRF2. The distinct error-prone mutational profile of fusion between critically shortened telomeres in human cells was reminiscent of Ku-independent microhomology-mediated end-joining.     

Ku complex controls the replication time of DNA in telomere regions

We have investigated whether the Ku complex is involved in regulating DNA replication in the yeast Saccharomyces cerevisiae. We find that Ku proteins control the replication time of telomeric regions; replication origins located close to telomeres or within subtelomeric repeat sequences normally initiate late, but are activated much earlier in mutants lacking Ku function. In contrast, origins distant from telomeres initiate replication at the normal time. Ku is one of the first components identified as important for replication timing, and specification of the replication time of chromosome ends by Ku is consistent with its role in maintaining telomere localization.     

Complete sequence of the genome of two dsRNA viruses from Discula destructiva

Complete nucleotide sequences were determined for the four dsRNA segments present in isolate 247 of Discula destructiva from South Carolina. The largest dsRNA (dsRNA 1) was 1787 bp in length with a single open reading frame (ORF) that coded for a putative RNA-dependent RNA polymerase (RdRp). The dsRNA 2 was 1585 bp in length with a single ORF that coded for a putative viral coat protein. Both the dsRNA 3 (1178 bp in length) and dsRNA 4 (308 bp) contained single ORFs. However, neither the nucleotide sequence nor the sequence of the putative translation products, showed any similarity with sequences currently available from GenBank. Although distinct, all 4 dsRNAs showed conserved nucleotides at both the 5′ and 3′ termini. Sequences of the two dsRNAs in an isolate of D. destructiva (331 originating from Idaho) were similar in length to, and shared similarity with, the dsRNA 1 and dsRNA 2 of isolate 247. However, although the putative RdRps of isolates 247 and 331 are closely related, the putative viral coat proteins coded for by the respective dsRNA 2s are distinct. Thus, the dsRNAs in the two fungal isolates appeared to originate from distinct, but related viruses, which we have named D. destructiva virus 1 and D. destructiva virus 2, respectively. Phylogenetic analysis indicated that the two viruses were most closely related to Fusarium solani virus 1 and should be considered members of the genus Partitivirus. Another isolate of D. destructiva (272.1) contains a 12 kb dsRNA in addition to the 4 dsRNAs found in isolate 247. Partial sequence of this 12 kb molecule showed a relationship to other large dsRNA molecules isolated from plants.     

DNA breaks are masked by multiple Rap1 binding in yeast: implications for telomere capping and telomerase regulation

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks by packaging them in a protective structure referred to as the telomere "cap." Here we investigate the nature of the telomere cap by examining events at DNA breaks generated adjacent to either natural telomeric sequences (TG repeats) or arrays of Rap1-binding sites that vary in length. Although DNA breaks adjacent to either short or long telomeric sequences are efficiently converted into stable telomeres, they elicit very different initial responses. Short telomeric sequences (80 base pair [bp]) are avidly bound by Mre11, as well as the telomere capping protein Cdc13 and telomerase enzyme, consistent with their rapid telomerase-dependent elongation. Surprisingly, little or no Mre11 binding is detected at long telomere tracts (250 bp), and this is correlated with reduced Cdc13 and telomerase binding. Consistent with these observations, ends with long telomere tracts undergo strongly reduced exonucleolytic resection and display limited binding by both Rpa1 and Mec1, suggesting that they fail to elicit a checkpoint response. Rap1 binding is required for end concealment at long tracts, but Rif proteins, yKu, and Cdc13 are not. These results shed light on the nature of the telomere cap and mechanisms that regulate telomerase access at chromosome ends.     

Repetitive sequences in the ribosomal intergenic spacer of Trypanosoma cruzi

A fragment of Trypanosoma cruzi ribosomal intergenic spacer (IGS) located at 6.7 kb from the 3′ end of the 24S rRNA gene was analyzed. This IGS fragment is characterized by the presence of three types of repetitive elements (designated Spacer Repetitive Elements, SRE), short direct repeats (5-6 bp) and chi-like recombinational sequences. SRE elements are composed of relatively short repeats (43–145 bp) which show variabilities consisting of nucleotide changes, insertions and deletions. SRE-1 element (145 bp) has a short oligo(dA) tail at the end of the repeat and can be found flanked by other SRE elements. SRE elements are species-specific, suggesting that probes based on them may be diagnostic for Trypanosoma cruzi.     

Stable inheritance of telomere chromatin structure and function in the absence of telomeric repeats

It is generally believed that telomeric repeats are a necessary and sufficient cis-element for telomere function. Here we show that telomere structure and meiotic function are stably inherited in fission yeast circular chromosomes that have lost all telomeric repeats. We found that the telomeric repeat binding protein, Taz1, and the heterochromatin protein, Swi6, remain associated with subtelomeres in the absence of telomeric repeats. We also found that the fusion point of circular chromosomes that lack telomeric repeats associates with SPB (the yeast counterpart of the centrosome) in the premeiotic horsetail stage, similarly to wild-type telomeres. However, a taz1+ deletion/reintroduction experiment revealed that the maintenance of Taz1 binding and premeiotic function is achieved via different strategies. Taz1 is recruited to subtelomeres by an autonomous element present in subtelomeric DNA, thus in a genetic mechanism. In contrast, the premeiotic subtelomere-SPB association is maintained in an epigenetic manner. These results shed light on the previously unrecognized role played by the subtelomere and underscore the robust nature of the functional telomere complex that is maintained by both genetic and epigenetic mechanisms. Furthermore, we suggest that the establishment and the maintenance of the functional telomere complex are mechanistically distinguishable.     

Aging-associated alteration of telomere length and subtelomeric status in female patients with Parkinson's disease

A telomere is a repetitive DNA structure at chromosomal ends that stabilizes the chromosome structure and prevents harmful end-to-end recombinations. The telomere length of somatic cells becomes shorter with aging because of the "end replication problem." This telomere shortening is accelerated by pathophysiological conditions including daily mental stress. Living with Parkinson's disease (PD) causes physical and mental stress; therefore, the authors hypothesized that the telomere length of somatic cells was shortened excessively in patients with PD. In order to detect PD-associated somatic telomeric alterations, the telomere length and subtelomeric methylation status of peripheral leukocytes of PD patients were assessed by Southern blotting, using methylation-sensitive and -insensitive isoschizomers. The results demonstrated that the peripheral leukocytes of Japanese female patients with PD bore fewer long telomeres and a proportional increase of hypomethylated subtelomeres in short telomeres in comparison with the healthy controls. This study indicates that with the neurodegeneration associated with PD, telomeric and subtelomeric structural alterations occur. These structural telomere alterations most likely occur secondary to the acceleration of aging-associated telomeric changes and the accelerated loss of cells bearing short telomeres.     

Aging-Associated Alteration of Telomere Length and Subtelomeric Status in Female Patients With Parkinson's Disease

Abstract: A telomere is a repetitive DNA structure at chromosomal ends that stabilizes the chromosome structure and prevents harmful end-to-end recombinations. The telomere length of somatic cells becomes shorter with aging because of the “end replication problem.” This telomere shortening is accelerated by pathophysiological conditions including daily mental stress. Living with Parkinson's disease (PD) causes physical and mental stress; therefore, the authors hypothesized that the telomere length of somatic cells was shortened excessively in patients with PD. In order to detect PD-associated somatic telomeric alterations, the telomere length and subtelomeric methylation status of peripheral leukocytes of PD patients were assessed by Southern blotting, using methylation-sensitive and -insensitive isoschizomers. The results demonstrated that the peripheral leukocytes of Japanese female patients with PD bore fewer long telomeres and a proportional increase of hypomethylated subtelomeres in short telomeres in comparison with the healthy controls. This study indicates that with the neurodegeneration associated with PD, telomeric and subtelomeric structural alterations occur. These structural telomere altertions most likely occur secondary to the acceleration of aging-associated telomeric changes and the accelerated loss of cells bearing short telomeres.     

Evolutionarily recent transfer of a group I mitochondrial intron to telomere regions in Saccharomyces cerevisiae

Summary The junctions between X and Y subtelomeric repeats in Saccharomyces cerevisiae usually contain a stretch of telomere sequences, (G_1–3T)_n. Two of three cloned X-Y junctions from strain YP1 have a replacement of about 200 bp of X, the internal telomere sequence, and 49 bp of Y by a 292 bp sequence. The first 227 bp of this insertion sequence are 100% identical to the fourth intron of cytochrome b. The rest of the insertion has homology to an unknown dispersed nuclear sequence. Recombination among subtelomeric regions can explain the nuclear distribution of this sequence and why telomeres can trap and maintain sequences that would otherwise be lost.     

Telomere length and telomerase activity: effect of ageing on human NK cells

Telomeres are repeats of TTAGGG sequences located at the end of eukaryotic chromosomes. They are essential for stabilisation and protection of chromosomal ends and for the regulation of cell replicative capacity. Due to the end-replication defect of DNA polymerase, telomeres shorten progressively with each cell division and telomere length may be an indicator of the replicative history of a cell. Compensatory mechanisms for the telomere loss have been identified. The most widely studied one is mediated by telomerase a ribonuclear protein-enzyme complex that synthesise telomeric repeats. In this study we have investigated whether NK cells, derived from a group of old healthy subjects, underwent the modifications of telomere length and telomerase activity observed in other sub-populations of lymphocytes with advancing age. We demonstrated that: (a) telomere shortening occurred and telomerase activity decreased in human NK cells with ageing; (b) the rate of telomere loss was different under and over 80 years of age; (c) similarly to telomere shortening, the modification of telomerase activity was particularly evident in octogenarians; (d) subjects with the most evident modifications of telomeres and telomerase were the oldest and those with increased NK cell numbers.