Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Telomeres cap chromosome ends and are pivotal for DNA stability. Deregulation of the telomere stabilising enzyme telomerase in malignancy has implications in diagnosis, prognosis and therapeutics of cancer. Quantification of the expression of the telomerase catalytic subunit, hTERT, using the LightCycler TeloTAGGG hTERT Quantification kit is not optimal for analysis of chronic myeloid leukemia (CML) samples. The internal control, porphobilinogen deaminase (PBGD) is amplified in a separate tube to hTERT and has an unstable genomic localisation of 11q23. Our laboratory thus developed a real-time reverse transcriptase polymerase chain reaction which co-amplifies hTERT and either mitochondrial single-stranded DNA binding protein 1 (ssBP1) or ubiquitin C (UBC).
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PMID:Real-time quantitative RT-PCR for human telomere elongation reverse transcriptase in chronic myeloid leukemia. 1523 74

Telomerase is a cellular reverse transcriptase that elongates the single-stranded chromosome ends and oligonucleotides in vivo and in vitro. In Saccharomyces cerevisiae, Est2p (telomerase catalytic subunit) and Tlc1 (telomerase RNA template subunit) constitute the telomerase core complex. We co-overexpressed GST (glutathione S-transferase)-Est2p and Tlc1 in S. cerevisiae, and reconstituted the telomerase activity. The GST-Est2p-Tlc1 complex was partially purified by ammonium sulphate fractionation and affinity chromatography on glutathione beads, and the partially purified telomerase did not contain the other two subunits of the telomerase holoenzyme, Est1p and Est3p. The purified recombinant GST-Est2p-Tlc1 telomerase core complex could specifically add nucleotides on to the single-stranded TG(1-3) primer in a processive manner, but could not translocate to synthesize more than one telomeric repeat. The purified telomerase core complex exhibited different activities when primers were paired with the Tlc1 template at different positions. The procedure of reconstitution and purification of telomerase core enzyme that we have developed now allows for further mechanistic studies of the functions of other subunits of the telomerase holoenzyme as well as other telomerase regulation proteins.
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PMID:Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast. 1581 5

Our project is designed to clone a 1.3kb gene fragment of telomerase catalytic subunit gene which contains seven reverse transcriptase motifs and specific region with conserved sequence termed "T motif". The gene fragment was amplified by PCR and was inserted into expression vector pET28-b. The recombinant plasmid was induced by IPTG for 4h and a 52KD recombinant protein was produced. Amount of hTRT recombinant protein expression was 20% of total bacterial protein in the form of inclusion. Inclusion was dissolved in 8 mol/L urea and 10 mmol/L DTT and carried out affinity purification under denaturing condition. The purified hTRT recombinant protein was conformed by Western-blot successfully.
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PMID:[Expression and purification of telomerase resever transcriptase motifs in E.coli]. 1597 68

Telomerase is a reverse transcriptase that maintains telomeres by adding telomeric TTAGGG repeats to the ends of human chromosomes. The aim of this study was to evaluate quantitatively the mRNA expression of telomerase catalytic subunit (hTERT) in different types of intracranial tumours in relation to their histologic pattern and grade and correlate it with progression-free (PFS) and overall survival (OS) of patients. Human telomerase reverse transcriptase mRNA levels were estimated by the use of real time RT-PCR in 68 samples of intracranial tumours. It revealed statistical correlation between hTERT mRNA expression levels and the grade of the tumours (P<0.001). Patients having negative expression of hTERT mRNA had statistically longer PFS (P=0.031) and OS (P=0.047). Cox univariate regression analysis revealed that hTERT mRNA-positive patients had a high and statistically significant risk of relapse (hazard ratio (HR) of 2.24 and P=0.038). In the Cox multivariate regression model, the levels of hTERT mRNA were adjusted for tumour grade and patients age, and since there was statistically significant relationship between the levels of hTERT mRNA and the grade of the tumours (P=0.003 or P=0.006, respectively), hTERT mRNA levels could not be considered as an independent prognostic factor for PFS or OS.
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PMID:mRNA quantification and clinical evaluation of telomerase reverse transcriptase subunit (hTERT) in intracranial tumours of patients in the island of Crete. 1598 35

Telomerase is a reverse transcriptase that maintains chromosome integrity through synthesis of repetitive telomeric sequences on the ends of eukaryotic chromosomes. In the yeast Saccharomyces cerevisiae, telomere length homeostasis is achieved through negative regulation of telomerase access to the chromosome terminus by telomere-bound Rap1 protein and its binding partners, Rif1p and Rif2p, and positive regulation by factors such as Ku70/80, Tel1p, and Cdc13p. Here we report the identification of mutations within an N-terminal region (region I) of the yeast telomerase catalytic subunit (Est2p) that cause telomere lengthening without altering measurable catalytic properties of the enzyme in vitro. These telomerase mutations affect telomere length through a Ku-independent mechanism and do not alter chromosome end structure. While Tel1p is required for expression of the telomere-lengthening phenotype, Rif1p and Rif2p are not, suggesting that telomere overextension is independent of Rap1p. Taken together, these data suggest that specific amino acids within region I of the catalytic subunit of yeast telomerase play a previously unanticipated role in the response to Tel1p regulation at the telomere.
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PMID:Regulation of telomere length by an N-terminal region of the yeast telomerase reverse transcriptase. 1619 86

Telomerase, a specialized reverse transcriptase that maintains telomere during cell division, is commonly associated with cell proliferation. Increasing evidence suggests that telomerase may bear functions other than telomere elongation. We investigated whether telomerase is expressed in the continuously growing goldfish retina. Telomeric repeat amplification protocol (TRAP) assay reveals telomerase activity in goldfish retina. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot show that telomerase catalytic subunit (TERT) is expressed at both mRNA and protein levels. Localization of TERT by immunohistochemistry indicates prominent expression of TERT in the outer nuclear layer, the inner nuclear layer, and, in a small population of cells, in the ganglion cell layer. Coexpression of TERT with proliferative cell nuclear antigen (PCNA) immunoreactivity is found in rod progenitor cells. These results suggest the role of telomerase in vertebrate central nervous system (CNS) other than telomere maintenance, such as regulation of cell cycle progression and maintenance of retinal cell phenotypes.
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PMID:Expression of telomerase reverse transcriptase in adult goldfish retina. 1787

When the telomerase catalytic subunit (Trt1/TERT) is deleted, a majority of fission yeast cells survives by circularizing chromosomes. Alternatively, a small minority survives by maintaining telomeric repeats through recombination among telomeres. The recombination-based telomere maintenance in trt1Delta cells is inhibited by the telomere protein Taz1. In addition, catalytically inactive full-length Trt1 (Trt1-CI) and truncated Trt1 lacking the T-motif and reverse transcriptase (RT) domain (Trt1-DeltaT/RT) can strongly inhibit recombination-based survival. Here, we investigated the effects of deleting the heterochromatin proteins Swi6 (HP1 ortholog) and Clr4 (Suv39 family of histone methyltransferases) and the telomere capping complex subunits Poz1 and Ccq1 on Taz1- and Trt1-dependent telomere recombination inhibition. The ability of Taz1 to inhibit telomere recombination did not require Swi6, Clr4, Poz1, or Ccq1. Although Swi6, Clr4, and Poz1 were dispensable for the inhibition of telomere recombination by Trt1-CI, Ccq1 was required for efficient telomere recruitment of Trt1 and Trt1-CI-dependent inhibition of telomere recombination. We also found that Swi6, Clr4, Ccq1, the checkpoint kinase Rad3 (ATR ortholog), and the telomerase regulatory subunit Est1 are all required for Trt1-DeltaT/RT to inhibit telomere recombination. However, because loss of Swi6, Clr4, Rad3, Ccq1, or Est1 did not significantly alter the recruitment efficiency of Trt1-DeltaT/RT to telomeres, these factors are likely to enhance the ability of Trt1-DeltaT/RT to inhibit recombination-based survival by contributing to the negative regulation of telomere recombination.
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PMID:Roles of heterochromatin and telomere proteins in regulation of fission yeast telomere recombination and telomerase recruitment. 2004 May 95

We previously demonstrated in prostate cancer cells that a phytochemical-perillyl alcohol-and the mechanistic target of rapamycin (mTOR) inhibitor rapamycin rapidly attenuated telomerase activity. Protein levels of the telomerase catalytic subunit reverse transcriptase (hTERT) were diminished in the absence of an effect on hTERT mRNA, supporting an effect on 4E-BP1 phosphorylation and reduced initiation of protein translation. The decline in hTERT protein did not coincide wholly, however, with loss of telomerase activity suggesting a further level of regulation. We hypothesized that a hTERT-mTOR-S6K (S6 kinase)-Hsp90 (Heat shock protein 90)-Akt complex previously detected in activated NK cells was present in DU145 prostate cancer cells. Furthermore, we postulated that both perillyl alcohol and rapamycin disrupted this complex to control telomerase activity post-translationally. Antibodies directed against either RAPTOR, a binding partner of mTOR, or mTOR itself co-immunoprecipitated Hsp90, hTERT, and S6K confirming a similar TERT complex in prostate cancer cells. Perillyl alcohol or rapamycin caused rapid dissociation of the captured hTERT-mTOR-RAPTOR complex, establishing an additional mechanism by which these agents decrease telomerase activity. These findings provide convincing evidence for mTOR-mediated regulation of hTERT in DU145 cells.
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PMID:Disruption of an hTERT-mTOR-RAPTOR protein complex by a phytochemical perillyl alcohol and rapamycin. 2328 42

In most human cancer cells, cellular immortalization relies on the activation and recruitment of telomerase to telomeres. The telomere-binding protein TPP1 and the TEN domain of the telomerase catalytic subunit TERT regulate telomerase recruitment. TERT contains a unique domain, called the insertion in fingers domain (IFD), located within the conserved reverse transcriptase domain. We report the role of specific hTERT IFD residues in the regulation of telomerase activity and processivity, recruitment to telomeres, and cell survival. One hTERT IFD variant, hTERT-L805A, with reduced activity and processivity showed impaired telomere association, which could be partially rescued by overexpression of TPP1-POT1. Another previously reported hTERT IFD mutant enzyme with similarly low levels of activity and processivity, hTERT-V791Y, displayed defects in telomere binding and was insensitive to TPP1-POT1 overexpression. Our results provide the first evidence that the IFD can mediate enzyme processivity and telomerase recruitment to telomeres in a TPP1-dependent manner. Moreover, unlike hTERT-V791Y, hTERT-V763S, a variant with reduced activity but increased processivity, and hTERT-L805A, could both immortalize limited-life-span cells, but cells expressing these two mutant enzymes displayed growth defects, increased apoptosis, DNA damage at telomeres, and short telomeres. Our results highlight the importance of the IFD in maintaining short telomeres and in cell survival.
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PMID:The Insertion in Fingers Domain in Human Telomerase Can Mediate Enzyme Processivity and Telomerase Recruitment to Telomeres in a TPP1-Dependent Manner. 2650 84


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