Complex and Tandem Repeat Structure of Subtelomeric Regions in the Taiwan Cricket, <Emphasis Type="BoldItalic">Teleogryllus taiwanemma</Emphasis>

Telomeres of most insects are composed of simple (TTAGG) _n repeats that are synthesized by telomerase. However, in some dipteran insects such as Drosophila melanogaster, (TTAGG) _n repeats or telomerase activity has not been detected. Although telomere structure is well documented in Diptera and Lepidoptera, very limited information is available on lower insect groups. To understand general aspects of telomere function and evolution in insects, we endeavored to characterize structures of the telomeric and subtelomeric regions in a lower insect, the Taiwan cricket, Teleogryllus taiwanemma. FISH analysis of this insect's chromosomes demonstrated (TTAGG) _n repeat elements in all distal ends. Just proximal to the telomeric repeats, the highly conserved 9-kb long terminal unit (LTU) sequences are tandemly repeated. These were observed in four of six chromosomes, three autosomal ends, and one X-chromosomal end. LTU sequences represent about 0.2% of the T. taiwanemma genome. Each LTU contains a core (TTAGG)₈-like sequence (TRLS) and five types of conserved sequences—ST (short telomere associated), J (joint), X, SR (satellite sequence rich), and Y—which vary in length from about 150 bp to 2.7 kb. The LTU sequence is defined as ST–J–TRLS–SR–X–Y–X–Y–X. Most LTU regions may be derived from the ancestral common sequence, which is observed in ST regions six times and at many other LTU sites. We could not find the LTU-like sequence in three other crickets including the closest species, T. emma, suggesting that the LTU in T. taiwanemma has been rapidly amplified in subtelomeric regions through recent evolutional events. It is also suggested that the highly conserved structure of the LTU is maintained by recombination and may contribute to telomere elongation, as seen in dipteran insects. Received: 6 August 2001/Accepted: 10 October 2001     

Phylogenetic distribution of TTAGG telomeric repeats in insects.

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

Characterization of TTAGG telomeric repeats,their interstitial occurrence and constitutively active telomerase in the mealybug <Emphasis Type="Italic">Planococcus lilacinus</Emphasis> (Homoptera; Coccoidea)

We confirmed the occurrence of the insect TTAGG telomeric repeats in the mealybug Planococcus lilacinus, a radiation-resistant coccid, by single primer polymerase chain reaction (PCR) and Southern hybridization. Analysis of Bal31 nuclease-digested DNA by Southern hybridization and chromosomes by FISH suggests that these repeats occur mainly at the ends of the chromosomes. However, sequence analysis of the PCR products of TTAGG-associated sequences from genomic DNA showed their interstitial occurrence and association with certain unrelated low-copy repeats. Because of their shorter length, the interstitial TTAGG sequences were detectable by primed in situ hybridizations but not by FISH. Analysis of chromosomes recovered after irradiation by fluorescent in situ hybridization suggested acquisition of TTAGG repeats at a majority of the healed ends. We also observed mild telomerase activity in unirradiated insects which was further enhanced after irradiation. Taken together, these results suggest that the mealybug has an efficient mechanism of formation of TTAGG repeats at radiation-induced chromosome ends and constitutively active telomerase may be a feature associated with rapid recovery of chromosome ends damaged by ionizing radiation.     

TCAGG,an alternative telomeric sequence in insects

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

Expression of telomerase activity is closely correlated with the capacity for cell division in tobacco plants

Telomerase is a specialized RNA-directed DNA polymerase that adds telomeric repeats to the ends of linear eukaryotic chromosomes. This activity is developmentally regulated in mammals. Here, we investigated the expression of telomerase activities in various cell types of tobacco plants using the telomere repeat amplification protocol (TRAP) assay. The greatest telomerase activity was detected in BY-2 suspension culture cells, while a relatively high level of activity was also found in roots. Because these two cell types contain a high proportion of actively dividing cells, our results indicate a close correlation between telomerase activity and the capacity for division in tobacco cells. Consistent with this observation was the very low level of telomerase activity in stems, leaves, and flowers, all tissues that had negligible activity of cell division. The specific expression of telomerase in actively dividing plant cells suggests that the pattern of telomerase regulation is largely conserved between higher plants and mammals.     

Telomerase activity and telomere detection during early bovine development

The ends of mammalian chromosomes are composed of repeated DNA sequences of (TTAGGG)(n) known as telomeres. Telomerase is a ribonucleoprotein that synthesizes telomeric DNA to replenish the 50-200 bp lost during cell replication. Cellular aging and senescence are associated with a lack of telomerase activity and a critical shortening of the telomere. The objectives of this study were to confirm the presence of TTAGGG repeats on the chromosomes of bovine embryos using in situ hybridization and assess the relative amounts of telomerase activity using a telomeric repeat amplification protocol (TRAP) during oocyte maturation and early embryo development. Applying a telomere DNA probe to the chromosomes of blastocysts and adult fibroblasts, telomeres were identified on the terminal ends of the p and q arms of chromosomes in all cells examined. Immature oocytes, matured oocytes, zygotes, 2- to 5-cell embryos, 6- to 8-cell embryos, morulae, and blastocysts were lysed in NP-40 lysis buffer and telomerase activity was assayed using the TRAP assay. Telomerase activity was detected in all developmental stages examined. Relative telomerase activity (based on telomerase internal standards and positive controls) appeared to decrease during oocyte maturation and subsequent development to the 8-cell stage but significantly increased (P < 0.05) by approximately 40-fold at the morula and blastocyst stages. It was concluded that the telomeres of bovine chromosomes contain TTAGGG repeats and that telomerase activity is up-regulated in morulae and blastocysts.     

Identification of a pentanucleotide telomeric sequence, (TTAGG)n, in the silkworm Bombyx mori and in other insects.

A pentanucleotide repetitive sequence, (TTAGG)n, has been isolated from a silkworm genomic library, using cross-hybridization with a (TTNGGG)5 sequence, which is conserved among most eukaryotic telomeres. Both fluorescent in situ hybridization and Bal 31 exonuclease experiments revealed major clusters of (TTAGG)n at the telomeres of all Bombyx chromosomes. To determine the evolutionary origin of this sequence, two types of telomeric sequence, (TTAGG)5 and a hexanucleotide repetitive sequence, (TTAGGG)4, which is conserved mainly among vertebrate and several invertebrate telomeres so far examined, were hybridized to DNAs from a wide variety of eukaryotic species under highly stringent hybridization conditions. The (TTAGGG)5 oligonucleotide hybridized to genomic DNAs from vertebrates and several nonvertebrate species, as has been reported so far, but not to any DNAs from insects. On the other hand, the Bombyx type of telomere sequence, (TTAGG)n, hybridized to DNAs from 8 of 11 orders of insect species tested but not to vertebrate DNAs, suggesting that this TTAGG repetitive sequence is conserved widely among insects.     

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.     

Ontogeny of telomerase in chicken: impact of downregulation on pre- and postnatal telomere length in vivo

Telomeres are the termini of linear chromosomes composed of tandem repeats of a conserved DNA sequence. Telomerase provides a mechanism for proliferating cells to offset telomeric sequence erosion by synthesizing new repeats onto the end of each parental DNA strand. Reduced or absent telomerase activity can lead to telomere shortening and genome instability. Telomeres and telomerase have not previously been characterized during ontogeny of any avian species. In the present study, telomerase activity in the chicken model was examined from early differentiation embryos through to adulthood. Telomerase activity was detected in all early embryos (preblastula through neurula) and in tissues throughout organogenesis. Subsequently, telomerase was downregulated in the majority of somatic tissues, either pre- or postnatally. A subset of tissues, such as intestine, immune and reproductive organs, exhibited constitutive activity. The impact of telomerase downregulation on telomere length was investigated and a telomere reduction of 3.2 kb in somatic tissues compared with germ line was observed in 5-year-old adults. The present results suggest that the telomere clock function is a conserved feature of avians as well as mammals. Knowledge regarding the relationships among telomerase regulation, proliferation/senescence profiles and differentiation status will be useful for numerous applications of chicken cells.     

Telomerase Activity and Telomere Length in Daphnia

Telomeres, comprised of short repetitive sequences, are essential for genome stability and have been studied in relation to cellular senescence and aging. Telomerase, the enzyme that adds telomeric repeats to chromosome ends, is essential for maintaining the overall telomere length. A lack of telomerase activity in mammalian somatic cells results in progressive shortening of telomeres with each cellular replication event. Mammals exhibit high rates of cell proliferation during embryonic and juvenile stages but very little somatic cell proliferation occurs during adult and senescent stages. The telomere hypothesis of cellular aging states that telomeres serve as an internal mitotic clock and telomere length erosion leads to cellular senescence and eventual cell death. In this report, we have examined telomerase activity, processivity, and telomere length in Daphnia, an organism that grows continuously throughout its life. Similar to insects, Daphnia telomeric repeat sequence was determined to be TTAGG and telomerase products with five-nucleotide periodicity were generated in the telomerase activity assay. We investigated telomerase function and telomere lengths in two closely related ecotypes of Daphnia with divergent lifespans, short-lived D. pulex and long-lived D. pulicaria. Our results indicate that there is no age-dependent decline in telomere length, telomerase activity, or processivity in short-lived D. pulex. On the contrary, a significant age dependent decline in telomere length, telomerase activity and processivity is observed during life span in long-lived D. pulicaria. While providing the first report on characterization of Daphnia telomeres and telomerase activity, our results also indicate that mechanisms other than telomere shortening may be responsible for the strikingly short life span of D. pulex.     

Telomerase activity is widespread in adult somatic tissues of Xenopus

Chromosome ends, or telomeres, are maintained by telomerase. Work in selected vertebrates has implied that telomerase is often repressed in differentiated cells, and telomere erosion results in senescence of cultured cells. Tissues from mature Xenopus laevis frogs were examined for telomerase enzymatic activity with the TRAP (telomere repeat amplification protocol) assay. All tissues contained active telomerase, most abundantly in testis, spleen, liver, and embryos; activity was less abundant but still readily detectable with < 100 ng of protein extract from brain and muscle tissues. Activity in somatic tissues of the diploid Xenopus tropicalis suggests this condition is not limited to the polyploid members of the genus, and that extensive differentiation-linked telomerase repression does not occur in Xenopus.     

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.Compared to the traditional TRAP assay that utilize ³²P labeled radioactive dCTP''s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.     

Developmental control of telomere lengths and telomerase activity in plants.

Telomere lengths and telomerase activity were studied during the development of a model dioecious plant, Melandrium album (syn Silene latifolia). Telomeric DNA consisted of Arabidopsis-type TTTAGGG tandem repeats. The terminal positions of these repeats were confirmed by both Bal31 exonuclease degradation and in situ hybridization. Analysis of terminal restriction fragments in different tissues and ontogenetic stages showed that telomere lengths are stabilized precisely and do not change during plant growth and development. Telomerase activity tested by using a semiquantitative telomerase repeat amplification protocol correlated with cell proliferation in the tissues analyzed. Highest activity was found in germinating seedlings and root tips, whereas we observed a 100-fold decrease in telomerase activity in leaves and no activity in quiescent seeds. Telomerase also was found in mature pollen grains. Telomerase activity in tissues containing dividing cells and telomere length stability during development suggest their precise control during plant ontogenesis; however, the telomere length regulation mechanism could be unbalanced during in vitro dedifferentiation.     

Techniques in plant telomere biology

The role model systems have played in understanding telomere biology has been enormous, and understanding has rapidly transferred to human telomere research. Most work using model organisms to study telomerase and nontelomerase-based telomere-maintenance systems has centered on yeasts, ciliates, and insects. But it is now timely to put considerably more effort into plant models for a number of reasons: (i) the rice and Arabidopsis genome sequencing projects make data mining possible; (ii) extensive collections of insertion mutants of Arabidopsis thaliana enable phenotypic effects of protein gene knockouts to be analyzed, including for those genes involved in telomere structure, function (including, for example, in meiosis), and maintenance; and (iii) the variability of plant telomeres is considerable and ranges from the telomerase-mediated synthesis of the Arabidopsis-type (TTTAGGG) and vertebrate-type (TTAGGG) repeats to sequences synthesized by telomerase-independent mechanism(s) that are still to be discovered. Here we describe how the understanding of telomere biology has been advanced by methods used to isolate telomeric sequences and prove that the putative sequences isolated are indeed telomeric. We show how assays designed to prove the activity of telomerase [e.g., telomeric repeat amplification protocol (TRAP)] lead not only to an understanding of telomere structure and function, but also to the understanding of cell activity in development and in the cell cycle. We review how assays designed to reveal protein/protein and protein/nucleic acid interactions promote understanding of the structure and activities of plant telomeres. Together, the data are making significant contributions to telomere biology in general and could have medical implications.     

Telomerase activity in human germline and embryonic tissues and cells

Telomerase is a ribonucleoprotein that synthesizes telomere repeats onto chromosome ends and is involved in maintaining telomere length in germline tissues and in immortal and cancer cells. In the present study, the temporal regulation of expression of telomerase activity was examined in human germline and somatic tissues and cells during development. Telomerase activity was detected in fetal, newborn, and adult testes and ovaries, but not in mature spermatozoa or oocytes. Blastocysts expressed high levels of telomerase activity as did most human somatic tissues at 16–20 weeks of development with the exception of human brain tissue. This activity could no longer be detected in the somatic tissues examined from the neonatal period onward. Neither placenta nor cultured fetal amniocytes contained detectable telomerase activity. Fetal tissues explanted into primary cell culture showed a dramatic decline in telomerase activity which became undetectable after the first passage in vitro. Elucidation of the regulatory pathways involved in the repression of telomerase activity during development may lead to the ability to manipulate telomerase levels and explore the consequences both for cellular aging and for the survival of cancer cells. © 1996 Wiley-Liss, Inc.     

The cytotoxic effects of single-stranded telomere mimics on OMA-BL1 cells.

Telomerase is a ribonucleoprotein that adds 5'-d(TTAGGG)-3' hexameric repeats onto the 3' ends of chromosomes. High telomerase activity has been associated with immortal cells, transformed cells, mitogenic stimulation, and proliferative diseases. It is not clear what phenotype would be observed by transient inhibition of telomerase. Studies were designed to inhibit telomerase activity using a series of S-ODN telomere sequence motifs. The studies evaluated the length, hydrogen bonding, and sequence requirements of telomerase inhibition using the TRAP assay and a bioassay measuring cell viability following exposure to the compounds. In addition, we have also studied the role of the 3' end and secondary structure of telomere mimics on telomerase inhibition. Observations reveal that sensitivity to the S-ODNs may not require hybridization to an antisense target but required guanine nucleotides on the 3' end for cells in culture and telomerase inhibition in vitro. The importance of H bonding and the requirement for a free 3' end for the activity of these compounds has also been demonstrated. However, transient inhibition of telomerase is not cytotoxic to all immortal cells and is not sufficient to explain the mechanism of cytotoxicity of these short oligonucleotides.