| Characterization of distinct and conserved features between ciliate and vertebrate telomerases | |
Abstract/OtherAbstract
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Telomeres are nucleoprotein structures that protect the ends of chromosomes from recombination and fusion. Telomeres are prefentially maintained by the enzyme telomerase. Telomerase is a reverse transcriptase, minimally composed of two core components, a catalytic subunit, TElomerase Reverse Transcriptase (TERT) and an RNA subunit, Telomerase RNA (TR) that carries the template for the replenishment of the telomeres. Telomerase is present throughout evolution, from ciliates and yeasts to vertebrates. Likely because it performs reverse transcription to maintain telomeres in these organisms, telomerases are remarkably conserved in terms of structure as well as in term of function. Nevertheless, some species-specific differences exist between telomerases from different model organisms. Studying these differences can deepen our knowledge of the telomerase enzyme, critical for human diseases such as cancer. The conservation and distinction between telomerases was explored by studying the functional conservation of a pseudoknot structure between Tetrahymena and human TRs and the response of mouse and human cells to the mutations of the subunits of telomerase. The hypothesis that the pseudoknot domains of the Tetrahymena and human TRs (tTR and hTR, respectively) may be functionally interchangeable was tested by constructing a chimeric TR (htTR) where the hTR pseudoknot domain was exchanged for tTR pseudoknot domain. htTR exhibited a weak, non processive telomerase activity in vitro, and was defective in telomere elongation, demonstrating that the Tetrahymena and human pseudoknots are not fully functionally interchangeable. Then, the importance of telomerase for cellular proliferation in mouse compared to human cells was investigated by studying the cellular consequences of mutating each core subunit of the mouse telomerase. First, the TERT-DN, a dominant-negative catalytically-inactive mutant of mTERT, was stably introduced into a mouse cell line (CB17) whose telomeres are 11kb to determine whether telomerase inhibition was telomere length-dependent in mouse cells. The CB17 cell line, in opposition to human cell lines with similar telomere length, rapidly reactivated telomerase, due to an increase in the level of expression of the endogenous mTERT. Second, a template-mutated mTR (mTR-muA) was stably introduced into a telomerase-positive (CB17) or a telomerase-negative cell line (DKO301). Compared to human telomerase-negative cells, DKO301-mTR muA displayed a delay in growth defects and eventual cell death, demonstrating that in this case and in the case of the mTERT-DN, mouse cells do not tolerate telomerase inhibition but can partly tolerate the expression of a template-mutated TR. Thus, despite conservation of diverse telomerases throughout evolution, we identified specific differences between the telomerases of ciliate and vertebrates and between the telomerases of human and mice. |
Authors
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Marie-Egyptienne, Delphine |
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Contributors
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Chantal Autexier (Supervisor) |
Publication Detail
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Publisher : McGill University Type : Electronic Thesis or Dissertation Format : application/pdf |
Date Detail
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2008 |
Subject
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Biology - Cell |
Coverage
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Relation
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Source
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Copyright Information
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© Delphine Marie-Egyptienne, 2008 |
Other Details
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Languages : en |
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