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Weiss-Schneeweiss H Riha K Jang CG Puizina J Scherthan H Schweizer D 《The Plant journal : for cell and molecular biology》2004,37(4):484-493
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. 相似文献
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Laura H. Bechard Bilge D. Butuner George J. Peterson Will McRae Zeki Topcu Michael J. McEachern 《Molecular and cellular biology》2009,29(3):626-639
Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent of human ALT cells. In this study, we generated telomeres composed of either of two types of mutant telomeric repeats, Acc and SnaB, that each alter the binding site for the telomeric protein Rap1. We show here that arrays of both types of mutant repeats present basally on a telomere were defective in negatively regulating telomere length in the presence of telomerase. Similarly, when each type of mutant repeat was spread to all chromosome ends in cells lacking telomerase, they led to the formation of telomeres produced by RTE that were much longer than those seen in cells with only wild-type telomeric repeats. The Acc repeats produced the more severe defect in both types of telomere maintenance, consistent with their more severe Rap1 binding defect. Curiously, although telomerase deletion mutants with telomeres composed of Acc repeats invariably showed extreme telomere elongation, they often also initially showed persistent very short telomeres with few or no Acc repeats. We suggest that these result from futile cycles of recombinational elongation and truncation of the Acc repeats from the telomeres. The presence of extensive 3′ overhangs at mutant telomeres suggests that Rap1 may normally be involved in controlling 5′ end degradation. 相似文献
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Using a modified single telomere length analysis protocol (STELA) to clone and examine the sequence composition of individual human XpYp telomeres, we discovered a distinct class of extremely short telomeres in human cancer cells with active telomerase. We name them "t-stumps," to distinguish them from the well-regulated longer bulk telomeres. T-stumps contained arrangements of telomeric repeat variants and a minimal run of seven canonical telomeric TTAGGG repeats, but all could bind at least one TRF1 or TRF2 in vitro. The abundance of t-stumps was unaffected by ATM alteration but could be changed by manipulating telomerase catalytic subunit (hTERT) levels in cancer cells. We propose that in the setting of active telomerase and compromised checkpoints characteristic of human cancer cells, t-stumps define the minimal telomeric unit that can still be protected by a TRF1/TRF2-capping complex and, further, that hTERT (or telomerase) may have a role in protecting t-stumps. 相似文献
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Telomere length in male germ cells is inversely correlated with telomerase activity 总被引:14,自引:0,他引:14
Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity. 相似文献
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Yeast mutants lacking telomerase are capable of maintaining telomeres by an alternate mechanism that depends on homologous recombination. We show here, by using Kluyveromyces lactis cells containing two types of telomeric repeats, that recombinational telomere elongation generates a repeating pattern common in most or all telomeres in survivors that retain both repeat types. We propose that these patterns arise from small circles of telomeric DNA being used as templates for rolling-circle gene conversion and that the sequence from the lengthened telomere is spread to other telomeres by additional, more typical gene conversion events. Consistent with this, artificially constructed circles of DNA containing telomeric repeats form long tandem arrays at telomeres when transformed into K. lactis cells. Mixing experiments done with two species of telomeric circles indicated that all of the integrated copies of the transforming sequence arise from a single original circular molecule. 相似文献
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Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles. 相似文献
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Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres
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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. 相似文献
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Recognition and elongation of telomeres by telomerase 总被引:9,自引:0,他引:9
Telomeres stabilize chromosomal ends and allow their complete replication in vivo. In diverse eukaryotes, the essential telomeric DNA sequence consists of variable numbers of tandem repeats of simple, G + C rich sequences, with a strong strand bias of G residues on the strand oriented 5' to 3' toward the chromosomal terminus. This strand forms a protruding 3' over-hang at the chromosomal terminus in three different eukaryotes analyzed. Analysis of yeast and protozoan telomeres showed that telomeres are dynamic structures in vivo, being acted on by shortening and lengthening activities. We previously identified and partially purified an enzymatic activity, telomere terminal transferase, or telomerase, from the ciliate Tetrahymena. Telomerase is a ribonucleoprotein enzyme with essential RNA and protein components. This activity adds repeats of the Tetrahymena telomeric sequence, TTGGGG, onto the 3' end of a single-stranded DNA primer consisting of a few repeats of the G-rich strand of known telomeric, and telomere-like, sequences. The shortest oligonucleotide active as a primer was the decamer G4T2G4. Structural analysis of synthetic DNA oligonucleotides that are active as primers showed that they all formed discrete intramolecular foldback structures at temperatures below 40 degrees C. Addition of TTGGGG repeats occurs one nucleotide at a time by de novo synthesis, which is not templated by the DNA primer. Up to 8000 nucleotides of G4T2 repeats were added to the primer in vitro. We discuss the implications of this finding for regulation of telomerase in vivo and a model for telomere elongation by telomerase. 相似文献
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Telomerase and differentiation in multicellular organisms: turn it off, turn it on, and turn it off again 总被引:19,自引:0,他引:19
Forsyth NR Wright WE Shay JW 《Differentiation; research in biological diversity》2002,69(4-5):188-197
Telomerase is a ribonucleoprotein complex that catalyses the addition of TTAGGG repeats onto telomeres, repetitive DNA structures found at the ends of linear chromosomes. The majority of human somatic tissues do not display telomerase activity and undergo telomeric shortening with consecutive divisions. This telomeric shortening results in replicative senescence in vitro and likely in vivo. Telomerase activity is present in the vast majority of tumors, preventing telomeric shortening and thereby enabling indefinite cell divisions. Telomerase activity is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onwards. However, regulated telomerase activity is seen in basal/stem cell compartments of highly regenerative tissues, such as those of the immune system, skin, and intestine. Avian species display telomerase repression and telomeric shortening similar to that seen in humans. However, rodents retain telomerase-competency throughout their lifespan and have not been shown to display division-dependent telomere shortening. The regulation of telomerase activity in plants is less well understood, although early indications suggest ubiquitous competency. The aim of this review is to present current data regarding developmental regulation of telomerase in humans, mice, chickens and flowering plants. Differentiation, quiescence and telomerase activity regulation will then be addressed in three human representative tissue systems; blood, skin, and intestine. We will also highlight similarities, differences and misconceptions in the developing field of telomere and telomerase biology. 相似文献
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Human POT1 facilitates telomere elongation by telomerase 总被引:39,自引:0,他引:39
Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase. 相似文献
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Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover. 相似文献
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Telomeres of most insects consist of pentanucleotide (TTAGG)n repeats, although the repeats are absent in Diptera and some other insect species, where the telomere regions are perhaps maintained without telomerase. To understand various and unusual telomere formation in insects, we have studied the characteristic features of a putative insect telomerase that has not been previously described. Using a modified telomeric repeat amplification protocol (TRAP), we first detected the telomerase activity in crickets, cockroaches and two Lepidopteran insects. The telomerase from crickets and cockroaches required dATP, dGTP and dTTP but not dCTP as a substrate and sequence analyses of the products of TRAP revealed that the (TTAGG)n repeats are synthesized by telomerase. The cockroach telomerase was detected both in somatic (fat body, muscle and neural tissues) and germ line (testis) cells, suggesting that expression of this enzyme is not regulated in a tissue-specific manner at an adult stage. While we detected high levels of telomerase activity in crickets and cockroaches, we could not detect activity in all tissues and cell cultures of the silkworm, Bombyx mori and in two Drosophila and one Sarcophaga cell lines. This supports the theory that Dipteran insects maintain their telomeres without telomerase. 相似文献
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Healing of a broken chromosome and in eukaryotes involves acquisition of a telomere. During macronuclear development in ciliated protozoans, germline chromosomes are fragmented into linear subchromosomes, whose ends are healed by de novo addition of telomeres. We showed previously that the ribonucleoprotein enzyme telomerase elongates preexisting telomeres by synthesizing one telomeric DNA strand, using a template sequence in the RNA moiety of the enzyme. By marking telomerase with a mutation in the telomerase RNA template, which causes synthesis of novel telomeric sequences, we now show that in the ciliate Tetrahymena, telomerase directly adds telomeric DNA onto nontelomeric sequences during developmentally controlled chromosome healing. Unexpectedly, one telomerase RNA template mutation converted telomerase from an enzyme that normally synthesizes precisely templated sequences to a less precise polymerase that sometimes synthesizes irregular telomeric repeats in vivo. 相似文献
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The telomeric nucleoprotein complex protects linear chromosome ends from degradation. In contrast to most eukaryotes in which telomerase is responsible for telomere elongation by adding short DNA repeats synthesized using an RNA template, the telomere elongation in Drosophila involves transposition of specialized telomeric retroelements onto chromosome ends. Proteins that bind telomeric and subtelomeric sequences form specific telomeric chromatin, and its components are highly conserved among organisms employing different mechanisms of telomere elongation. This review is focused on the analysis of components of the Drosophila telomeric complex and its comparison with telomeric proteins in telomerase-encoded organisms. Structural and functional analysis of Drosophila telomeres suggests that there are three distinct chromatin regions: protective structure at the very end of chromosome (cap), subtelomeric region which is characterized by condensed chromatin structure, and the terminal retrotransposon array whose expression is under the control of an RNAi (RNA interference)-based mechanism. The link between RNAi and telomeric chromatin formation in germinal tissues is discussed. 相似文献