Boundary elements of the Tetrahymena telomerase RNA template and alignment domains

Telomerase is a DNA polymerase fundamental to the replication and maintenance of telomere sequences at chromosome ends. The RNA component of telomerase is essential for the synthesis of telomere repeats. In vitro, the template domain (5'-CAACCCCAA-3') of the Tetrahymena telomerase RNA dictates the addition of Tetrahymena-specific telomere repeats d(TTGGGG)n, onto the 3' end of G-rich or telomeric substrates that are base-paired with the template and alignment regions of the RNA. Using a reconstituted in vitro system, we determined that altering the sequence of the alignment and template domains affects processivity of telomerase without abolishing telomerase activity. These results suggest that alternative template/alignment regions may be functional. In the ciliate telomerase RNAs, there is a conserved sequence 5'-(CU)GUCA-3', located two residues upstream of the template domain. The location and sequence of this conserved domain defined the 5' boundary of the template region. These data provide insights into the regulation of telomere synthesis by telomerase

Tetrahymena telomerase catalyzes nucleolytic cleavage and nonprocessive elongation

Telomerase is a ribonucleoprotein enzyme that adds telomeric repeats to chromosomes, maintaining telomere length and stabilizing chromosome ends. In vitro, telomerase from the ciliate Tetrahymena elongates single-stranded, guanosine-rich DNA primers by adding repeats of the Tetrahymena telomeric sequence, dT2G4. We have identified two activities of Tetrahymena telomerase in addition to the previously described processive elongation reaction: a 3'-5' nucleolytic cleavage of primer or product DNA and a nonprocessive mode of elongation. The nucleolytic cleavage activity removed residues not conforming to the telomeric repeat sequence from a primer 3' end, eliminating mismatch between DNA primer and RNA template sequences. Template-matched residues were also cleaved from primer or product DNA. Specific primer lengths, sequences, and concentrations stimulated cleavage and processive or nonprocessive elongation differentially. These newly identified activities suggest that telomerase may catalyze a range of telomere synthesis and repair functions and suggest mechanistic similarities between telomerase and RNA polymerase enzymes. On the basis of our results, we propose a model for telomerase primer binding, cleavage, and elongation

Leadership, the logic of sufficiency and the sustainability of education

The notion of sufficiency has not yet entered mainstream educational thinking, and it still has to make its mark upon educational leadership. However, a number of related concepts – particularly those of sustainability and complexity theory – are beginning to be noticed. This article examines these two concepts and uses them to critique the quasi-economic notion of efficiency, before arguing that the concept of sufficiency arises naturally from this discussion. This concept, originally derived from environmental thinking, has both metaphorical and practical impact for educational organizations and their leadership. An examination of three possible meanings suggests that while an embrace of an imperative concept of sufficiency seems increasingly necessary, its adoption would probably lead to a number of other problems, as it challenges some fundamental societal values and assumptions. Nevertheless, the article argues that these need to be addressed for the sake of both sustainable leadership and a sustainable planet

Extreme Telomere Length Dimorphism in the Tasmanian Devil and Related Marsupials Suggests Parental Control of Telomere Length

Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species. Here we report that animals in the Order Dasyuromorphia have chromosomes characterised by striking telomere length dimorphism between homologues. Findings in sex chromosomes suggest that telomere length dimorphism may be regulated by events in the parental germlines. Long telomeres on the Y chromosome imply that telomere lengthening occurs during spermatogenesis, whereas telomere diminution occurs during oogenesis. Although found in several somatic cell tissue types, telomere length dimorphism was not found in DFTD cancer cells, which are characterised by uniformly short telomeres. This is, to our knowledge, the first report of naturally occurring telomere length dimorphism in any species and suggests a novel strategy of telomere length control. Comparative studies in five distantly related marsupials and a monotreme indicate that telomere dimorphism evolved at least 50 million years ago. © 2012 Bender et al

Human Telomerase Reverse Transcriptase (hTERT) Q169 Is Essential for Telomerase Function In Vitro and In Vivo

BACKGROUND:Telomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS:To understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function. CONCLUSIONS/SIGNIFICANCE:We provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention

Angular Dependence of Neutrino Flux in KM3 Detectors in Low Scale Gravity Models

Cubic kilometer neutrino telescopes are capable of probing fundamental questions of ultra-high energy neutrino interactions. There is currently great interest in neutrino interactions caused by low-scale, extra dimension models. Above 1 PeV the cross section in low scale gravity models rises well above the total Standard Model cross section. We assess the observability of this effect in the 1 PeV - 100 PeV energy range of kilometer-scale detectors with several new points of emphasis that hinge on enhanced neutral current cross sections. A major point is the importance of ``feed-down'' regeneration of upward neutrino flux, driven by new-physics neutral current interactions in the flux evolution equations. Feed-down is far from negligible, and it is essential to include its effect. We then find that the angular distribution of events has high discriminating value in separating models. In particular the ``up-to-down'' ratio between upward and downward-moving neutrino fluxes is a practical diagnostic tool which can discriminate between models in the near future. The slope of the angular distribution, in the region of maximum detected flux, is also substantially different in low-scale gravity and the Standard Model. These observables are only weakly dependent on astrophysical flux uncertainties. We conclude that angular distributions can reveal a breakdown of the Standard Model and probe the new physics beyond, as soon as data become available.Comment: 25 pages, 6 figures, discussion of calculations expanded, references adde

Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-seq Protocol

Chemical mapping methods probe RNA structure by revealing and leveraging correlations of a nucleotide's structural accessibility or flexibility with its reactivity to various chemical probes. Pioneering work by Lucks and colleagues has expanded this method to probe hundreds of molecules at once on an Illumina sequencing platform, obviating the use of slab gels or capillary electrophoresis on one molecule at a time. Here, we describe optimizations to this method from our lab, resulting in the MAP-seq protocol (Multiplexed Accessibility Probing read out through sequencing), version 1.0. The protocol permits the quantitative probing of thousands of RNAs at once, by several chemical modification reagents, on the time scale of a day using a table-top Illumina machine. This method and a software package MAPseeker (http://simtk.org/home/map_seeker) address several potential sources of bias, by eliminating PCR steps, improving ligation efficiencies of ssDNA adapters, and avoiding problematic heuristics in prior algorithms. We hope that the step-by-step description of MAP-seq 1.0 will help other RNA mapping laboratories to transition from electrophoretic to next-generation sequencing methods and to further reduce the turnaround time and any remaining biases of the protocol.Comment: 22 pages, 5 figure

Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

BACKGROUND: Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. METHODS: The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. RESULTS: The Bcl-2 protein expression was found to be decreased in 105 (42%) cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089) associated with a worse disease free survival (DFS), while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. CONCLUSION: The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer

Telomerase and breast cancer

Current therapies for breast cancer include treatments that are toxic and often result in drug resistance. Telomerase, a cellular reverse transcriptase that maintains the ends of chromosomes (telomeres), is activated in the vast majority of breast cancers (over 90% of breast carcinomas) but not in normal adjacent tissues. Telomerase is thus an attractive target for both diagnosis and therapy because of its distinct pattern of expression. We address the use of telomerase in the diagnostics of breast pathology, as well as the use of telomerase inhibitors in the treatment and prevention of breast cancer