Comparative analysis of telomeric restriction fragment lengths in different tissues of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> trees of different age

Ginkgo trees of four different ages were selected as experimental material. Telomeric restriction fragment (TRF) lengths, as an indicator of telomere length, were determined for different tissues by Southern hybridization analysis. Statistical analysis was performed to compare two aspects of TRF length. By determining TRF lengths for different tissues for each age, a latent tendency was found. TRF length varied from short to long in these tissues in the order microspore < embryonal callus < leaf < branchlet. TRF lengths for leaf tissue and branchlet tissue were dissimilar for female and male mature trees, although this difference between TRF lengths for the two sexes was not statistically significant. Evaluation of TRF lengths for each tissue for trees of all four ages revealed TRF lengths increased with age to some extent. Different rates of change were found for leaf tissue and for branchlet tissue, although tendencies to increase were not linear for either. Finally, a simple mathematical model was formulated to describe the relationship between telomere length and age for Ginkgo biloba L.     

Change of season-specific telomere lengths in <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

Telomeres have lately received considerable attention in the development of tree species. Normal somatic cells have limited replicative capacity and telomeres get shorten with each round of DNA replication. For broad-leaved tree species, to determine what changes happen to their somatic cells in its annual development cycle, an exhaustive research on different ages of gingko trees telomere length changes was carried out. Analysis of changes in leaf telomere lengths in the annual development cycle of Ginkgo biloba L. showed no significant changes (P > 0.05) from April to August, but a dramatic decrease in September and October (P < 0.05). Statistical analyses showed that TRF length of males and females are equal, the p values of the three age groups comparison were all bigger than 0.05. The results showed that specific apoptotic changes occur in the annual development cycle of Ginkgo biloba L.     

Oxidative stress‐induced renal telomere shortening as a mechanism of cyclosporine‐induced nephrotoxicity

Due to the association of oxidative stress and telomere shortening, it was aimed in the present study to investigate the possibility whether cyclosporine‐A exerts its nephrotoxic side effects via induction of oxidative stress‐induced renal telomere shortening and senescent phenotype in renal tissues of rats. Renal oxidative stress markers, 8‐hydroxydeoxyguanosine, malondialdehyde, and protein carbonyl groups were measured by standard methods. Telomere length and telomerase activity were also evaluated in kidney tissue samples. Results showed that cyclosporine‐A treatment significantly (P < 0.05) enhanced renal malondialdehyde, 8‐hydroxydeoxyguanosine, and protein carbonyl groups levels, decreased renal telomere length, and deteriorated renal function compared with the controls. Renal telomerase activity was not affected by cyclosporine‐A. Renal telomere length could be considered as an important parameter of both oxidative stress and kidney function. Telomere shortening and accelerated kidney aging may be caused by cyclosporine‐induced oxidative stress, indicating the potential mechanism of cyclosporine‐induced nephrotoxicity.     

Characterisation of an unusual telomere motif (TTTTTTAGGG)n in the plant Cestrum elegans (Solanaceae), a species with a large genome

The characterization of unusual telomere sequence sheds light on patterns of telomere evolution, maintenance and function. Plant species from the closely related genera Cestrum, Vestia and Sessea (family Solanaceae) lack known plant telomeric sequences. Here we characterize the telomere of Cestrum elegans, work that was a challenge because of its large genome size and few chromosomes (1C 9.76 pg; n = 8). We developed an approach that combines BAL31 digestion, which digests DNA from the ends and chromosome breaks, with next‐generation sequencing (NGS), to generate data analysed in RepeatExplorer, designed for de novo repeats identification and quantification. We identify an unique repeat motif (TTTTTTAGGG)_n in C. elegans, occurring in ca. 30 400 copies per haploid genome, averaging ca. 1900 copies per telomere, and synthesized by telomerase. We demonstrate that the motif is synthesized by telomerase. The occurrence of an unusual eukaryote (TTTTTTAGGG)_n telomeric motif in C. elegans represents a switch in motif from the ‘typical’ angiosperm telomere (TTTAGGG)_n. That switch may have happened with the divergence of Cestrum, Sessea and Vestia. The shift in motif when it arose would have had profound effects on telomere activity. Thus our finding provides a unique handle to study how telomerase and telomeres responded to genetic change, studies that will shed more light on telomere function.     

Plant cells express telomerase activity upon transfer to callus culture, without extensively changing telomere lengths

Changes in telomere lengths and telomerase activity in tobacco cells were studied during dedifferentiation and differentiation; leaf tissues were used to initiate callus cultures, which were then induced to regenerate plants. While no significant changes in the range of telomere lengths were observed in response to dedifferentiation and differentiation, there was a conspicuous increase in telomerase activity in calli compared to the source leaves, where the activity was hardly detectable. In leaves of regenerated plants, the telomerase activity fell to almost the same level as in the original plant, showing on the average 0.04% of the level in callus. The process was then repeated using the regenerants as the source material. In the second round of dedifferentiation and differentiation, telomerase activity showed a similar increase in calli derived from regenerated plants and a drop in plants regenerated from these calli. Telomere lengths remained unchanged both in calli and in leaves of regenerants. The conservation of telomere lengths over repeated rounds of dedifferentiation and differentiation, which are associated with dramatic changes in cell division rate and corresponding variation in telomerase activity may reflect the function of a regulatory mechanism in plant cells which controls telomerase action to compensate for replicative loss of telomeric DNA. Received: 31 July 1998 / Accepted: 21 September 1998     

Expression of Telomerase and Telomere Length Are Unaffected by either Age or Limb Regeneration in Danio rerio

Background

The zebrafish is an increasingly popular model for studying many aspects of biology. Recently, ztert, the zebrafish homolog of the mammalian telomerase gene has been cloned and sequenced. In contrast to humans, it has been shown that the zebrafish maintains telomerase activity for much of its adult life and has remarkable regenerative capacity. To date, there has been no longitudinal study to assess whether this retention of telomerase activity equates to the retention of chromosome telomere length through adulthood.

Methodology/Principal Findings

We have systematically analyzed individual organs of zebrafish with regard to both telomere length and telomerase activity at various time points in its adult life. Heart, gills, kidney, spleen, liver, and intestine were evaluated at 3 months, 6 months, 9 months, and 2 years of age by Southern blot analysis. We found that telomeres do not appreciably shorten throughout the lifespan of the zebrafish in any organ. In addition, there was little difference in telomere lengths between organs. Even when cells were under the highest pressure to divide after fin-clipping experiments, telomere length was unaffected. All aged (2 year old) tissues examined also expressed active amounts of telomerase activity as assessed by TRAP assay.

Conclusions/Significance

In contrast to several other species including humans, the retention of lifelong telomerase and telomeres, as we have reported here, would be necessary in the zebrafish to maintain its tremendous regenerative capacity. The ongoing study of the zebrafish''s ability to maintain telomerase activity may be helpful in unraveling the complexity involved in the maintenance (or lack thereof) of telomeres in other species such the mouse or human.     

Tissue context-activated telomerase in human epidermis correlates with little age-dependent telomere loss

Telomerase- and telomere length regulation in normal human tissues is still poorly understood. We show here that telomerase is expressed in the epidermis in situ independent of age but was repressed upon the passaging of keratinocytes in monolayer culture. However, when keratinocytes were grown in organotypic cultures (OTCs), telomerase was re-established, indicating that telomerase activity is not merely proliferation-associated but is regulated in a tissue context-dependent manner in human keratinocytes. While not inducible by growth factors, treatment with the histone deacetylation inhibitor FK228 restored telomerase activity in keratinocytes grown in monolayer cultures. Accordingly, CHIP analyses demonstrated an acetylated, active hTERT promoter in the epidermis in situ and in the epidermis of OTCs but a deacetylated, silenced hTERT promoter with subsequent propagation in monolayer culture suggesting that histone acetylation is part of the regulatory program to guarantee hTERT expression/telomerase activity in the epidermis. In agreement with the loss of telomerase activity, telomeres shortened during continuous propagation in monolayer culture by an average of ～ 70 base pairs (bp) per population doubling (pd). However, telomere erosion varied strongly between different keratinocyte strains and even between individual cells within the same culture, thereby arguing against a defined rate of telomere loss per replication cycle. In the epidermis in situ, as determined from early-passage keratinocytes and tissue sections from different age donors, we calculated a telomere loss of only ～ 25 bp per year. Since we determined the same rate for the non-regenerating melanocytes and dermal fibroblasts, our data suggest that in human epidermis telomerase is a protective mechanism against excessive telomere loss during the life-long regeneration.     

Replication Protein A‐1 Has a Preference for the Telomeric G‐rich Sequence in Trypanosoma cruzi

Replication protein A (RPA), the major eukaryotic single‐stranded binding protein, is a heterotrimeric complex formed by RPA‐1, RPA‐2, and RPA‐3. RPA is a fundamental player in replication, repair, recombination, and checkpoint signaling. In addition, increasing evidences have been adding functions to RPA in telomere maintenance, such as interaction with telomerase to facilitate its activity and also involvement in telomere capping in some conditions. Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoa parasite that appears early in the evolution of eukaryotes. Recently, we have showed that T. cruziRPA presents canonical functions being involved with DNA replication and DNA damage response. Here, we found by FISH/IF assays that T. cruziRPA localizes at telomeres even outside replication (S) phase. In vitro analysis showed that one telomeric repeat is sufficient to bind RPA‐1. Telomeric DNA induces different secondary structural modifications on RPA‐1 in comparison with other types of DNA. In addition, RPA‐1 presents a higher affinity for telomeric sequence compared to randomic sequence, suggesting that RPA may play specific roles in T. cruzi telomeric region.     

The plant Pontin and Reptin homologues,RuvBL1 and RuvBL2a,colocalize with TERT and TRB proteins in vivo,and participate in telomerase biogenesis

Telomerase maturation and recruitment to telomeres is regulated by several telomerase‐ and telomere‐associated proteins. Among a number of proteins, human Pontin and Reptin play critical roles in telomerase biogenesis. Here we characterized plant orthologues of Pontin and Reptin, RuvBL1 and RuvBL2a, respectively, and show association of Arabidopsis thaliana RuvBL1 (AtRuvBL1) with the catalytic subunit of telomerase (AtTERT) in the nucleolus in vivo. In contrast to mammals, interactions between AtTERT and AtRuvBL proteins in A. thaliana are not direct and they are rather mediated by one of the Arabidopsis thaliana Telomere Repeat Binding (AtTRB) proteins. We further show that plant orthologue of dyskerin, named AtCBF5, is indirectly associated with AtTRB proteins but not with the AtRuvBL proteins in the plant nucleus/nucleolus, and interacts with the Protection of telomere 1 (AtPOT1a) in the nucleolus or cytoplasmic foci. Our genome‐wide phylogenetic analyses identify orthologues in RuvBL protein family within the plant kingdom. Dysfunction of AtRuvBL genes in heterozygous T‐DNA insertion A. thaliana mutants results in reduced telomerase activity and indicate the involvement of AtRuvBL in plant telomerase biogenesis.     

Dissociation of Telomere Dynamics from Telomerase Activity in Human Thyroid Cancer Cells

Prevention of telomere erosion through acquisition of telomerase activity is thought to be an essential mechanism in most human cancer cells for avoidance of cellular senescence and crisis. It has been generally assumed that once telomerase has been activated, no further telomere shortening should ensue. We show here, however, that a much more complex pattern of telomere dynamics can exist in telomerase-positive immortal cancer cells. Using a panel of subclones derived from a human thyroid cancer cell line, K1E7, we found that some clones show persistent decline in mean telomere restriction fragment (TRF) length by up to 2 kb over 450 population doublings (pd), despite sustained high telomerase activity (as assessed by thein vitro“TRAP” assay). TRF length subsequently stabilized at around 5 kb, but with no corresponding increase in telomerase activity. One clone showed an even more unexpected biphasic time course, with the mean TRF length initially increasing by 1.5 kb over 90 pd, before “plateauing” and then returning over a similar period to its original value, again without any correlation to TRAP activity. Such dissociations between telomere dynamics and telomerase activity support the existence of additional controls on telomere length in the intact cell. Our observations are consistent with current negative-feedback models of telomere length regulation by telomere binding proteins and these cell lines should prove useful experimental tools for their further evaluation.     

Telomere Length Dynamics in Telomerase-Positive Immortal Human Cell Populations

It has been proposed that the progressive shortening of telomeres in somatic cells eventually results in senescence. Previous experiments have demonstrated that many immortal cell lines have acquired telomerase activity leading to stabilization of telomere length. Telomere dynamics and telomerase activity were examined in the telomerase-positive immortal cell lines HeLa and 293 and subclones derived from them. A mass culture of HeLa cells had a stable mean telomere length over 60 population doublings (PD)in vitro.Subclones of this culture, however, had a range of mean telomere lengths indicating that telomeric heterogeneity exists within a population with a stable mean telomere length. Some of the subclones lacked detectable telomerase activity soon after isolation but regained it by PD 18, suggesting that at least some of the variation in telomere length can be attributed to variations in telomerase activity levels. 293 subclones also varied in telomere length and telomerase activity. Some telomerase-positive 293 subclones contained long telomeres that gradually shortened, demonstrating that factors other than telomerase also act to modulate telomere length. Fluctuations in telomere length in telomerase-positive immortalized cells may contribute to chromosomal instability and clonal evolution.     

TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site

Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G‐rich sequence, and with noncoding RNA, Telomeric repeat‐containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2′‐OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G‐quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate – the 3′‐end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3′‐end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Individual variation in early‐life telomere length and survival in a wild mammal

Individual variation in survival probability due to differential responses to early‐life environmental conditions is important in the evolution of life histories and senescence. A biomarker allowing quantification of such individual variation, and which links early‐life environmental conditions with survival by providing a measure of conditions experienced, is telomere length. Here, we examined telomere dynamics among 24 cohorts of European badgers (Meles meles). We found a complex cross‐sectional relationship between telomere length and age, with no apparent loss over the first 29 months, but with both decreases and increases in telomere length at older ages. Overall, we found low within‐individual consistency in telomere length across individual lifetimes. Importantly, we also observed increases in telomere length within individuals, which could not be explained by measurement error alone. We found no significant sex differences in telomere length, and provide evidence that early‐life telomere length predicts lifespan. However, while early‐life telomere length predicted survival to adulthood (≥1 year old), early‐life telomere length did not predict adult survival probability. Furthermore, adult telomere length did not predict survival to the subsequent year. These results show that the relationship between early‐life telomere length and lifespan was driven by conditions in early‐life, where early‐life telomere length varied strongly among cohorts. Our data provide evidence for associations between early‐life telomere length and individual life history, and highlight the dynamics of telomere length across individual lifetimes due to individuals experiencing different early‐life environments.     

Production of embryogenic callus from nonembryonic explants of Norway Spruce Picea abies (L.) Karst

Callus has been induced on needle and bud explants from 2, 6 and 26 year old Picea abies (L.) Karst, trees. Seventy seven percent of the needle explants from the youngest material was productive compared to 0.2% for the old tree. New embryogenic sections appeared in the non-embryogenic callus derived from 2, 6 and 26 year old trees after prolonged culture and was promoted by the use of an established embryogenic callus line in a nurse culture system.Abbreviations EDTA Ethylenediaminetetraacetic acid - RAPD Random amplified polymorphic DNA     

Intraspecific competition and water use efficiency in <Emphasis Type="Italic">Quercus suber</Emphasis>: evidence of an optimum tree density?

The dehesa and montado agroecosystems seem to be water-stress induced structures and some authors have found evidence of a relationship between stand tree density and mean annual precipitation. In order to assess the ecophysiological responses of Quercus suber to increasing tree density and to evaluate if there was evidence of an “optimum tree density” with respect to mean annual rainfall in our study area, we established a tree-density gradient of 20, 40, and 60 trees ha⁻¹, coded D20, D40, and D60, respectively. Trees in D40 plots had higher specific leaf area (SLA) values and the highest water content. Both D20 and D60 trees had leaves with higher dry matter content (LDMC). The trade-off between SLA and LDMC was very strong and placed D40 trees as the least stressed, in terms of leaf anatomy. We also found differences in mean carbon isotope discrimination (Δ), between different density plots, as big as 1.25‰. Again, D40 trees discriminated more against ¹³C than the trees from D20 and D60 plots, which suggests a less conservative water use strategy, possibly deriving from greater water availability. The lower water retention in the D20 areas and the increased competition for water resources in D60 plots placed trees, in both areas, in a more stressful situation, regarding water availability. The D40 areas thus seemed to represent a more favourable environment for Q. suber in our montado. Therefore, the areas with 40 trees ha⁻¹ suggest the existence of an optimum tree density with respect to annual rainfall in our site, and may represent a compromise between an increased retention of water resources and intraspecific competition.     

Enforced telomere elongation increases the sensitivity of human tumour cells to ionizing radiation

More than 85% of all human cancers possess the ability to maintain chromosome ends, or telomeres, by virtue of telomerase activity. Loss of functional telomeres is incompatible with survival, and telomerase inhibition has been established in several model systems to be a tractable target for cancer therapy. As human tumour cells typically maintain short equilibrium telomere lengths, we wondered if enforced telomere elongation would positively or negatively impact cell survival. We found that telomere elongation beyond a certain length significantly decreased cell clonogenic survival after gamma irradiation. Susceptibility to irradiation was dosage-dependent and increased at telomere lengths exceeding 17 kbp despite the fact that all chromosome ends retained telomeric DNA. These data suggest that an optimal telomere length may promote human cancer cell survival in the presence of genotoxic stress.