scholarly journals Plasmodium falciparum Telomerase: De Novo Telomere Addition to Telomeric and Nontelomeric Sequences and Role in Chromosome Healing

1998 ◽  
Vol 18 (2) ◽  
pp. 919-925 ◽  
Author(s):  
Emmanuel Bottius ◽  
Nassera Bakhsis ◽  
Artur Scherf
Keyword(s):  
Plasmodium Falciparum ◽  
Reverse Transcriptase ◽  
De Novo ◽  
Telomerase Activity ◽  
Telomeric Dna ◽  
Oligonucleotide Primers ◽  
Cell Extracts ◽  
Linear Chromosomes ◽  
Dna Primers

ABSTRACT Telomerase, a specialized cellular reverse transcriptase, compensates for chromosome shortening during the proliferation of most eucaryotic cells and contributes to cellular immortalization. The mechanism used by the single-celled protozoan malaria parasitePlasmodium falciparum to complete the replication of its linear chromosomes is currently unknown. In this study, telomerase activity has for the first time been identified in cell extracts ofP. falciparum. The de novo synthesis of highly variable telomere repeats to the 3′ end of DNA oligonucleotide primers by plasmodial telomerase is demonstrated. Permutated telomeric DNA primers are extended by the addition of the next correct base. In addition to elongating preexisting telomere sequences, P. falciparumtelomerase can also add telomere repeats onto nontelomeric 3′ ends. The sequence GGGTT… was the predominant initial DNA sequence added to the nontelomeric 3′ ends in vitro. Poly(C) at the 3′ end of the oligonucleotide significantly alters the precision of the new telomerase added repeats. The efficiency of nontelomeric primer elongation was dependent on the presence of a G-rich cassette upstream of the 3′ terminus. Oligonucleotide primers based on natural P. falciparum chromosome breakpoints are efficiently used as telomerase substrates. These results imply that P. falciparum telomerase contributes to chromosome maintenance and to de novo telomere formation on broken chromosomes. Reverse transcriptase inhibitors such as dideoxy GTP efficiently inhibitP. falciparum telomerase activity in vitro. These data point to malaria telomerase as a new target for the development of drugs that could induce parasite cell senescence.

scholarly journals Structural conservation in the template/pseudoknot domain of vertebrate telomerase RNA from teleost fish to human

2016 ◽  
Vol 113 (35) ◽  
pp. E5125-E5134 ◽  
Author(s):  
Yaqiang Wang ◽  
Joseph D. Yesselman ◽  
Qi Zhang ◽  
Mijeong Kang ◽  
Juli Feigon
Keyword(s):  
Reverse Transcriptase ◽  
Teleost Fish ◽  
Telomerase Activity ◽  
Full Length ◽  
Telomerase Rna ◽  
Dna Repeats ◽  
Base Pairs ◽  
Tertiary Interactions ◽  
Linear Chromosomes

Telomerase is an RNA–protein complex that includes a unique reverse transcriptase that catalyzes the addition of single-stranded telomere DNA repeats onto the 3′ ends of linear chromosomes using an integral telomerase RNA (TR) template. Vertebrate TR contains the template/pseudoknot (t/PK) and CR4/5 domains required for telomerase activity in vitro. All vertebrate pseudoknots include two subdomains: P2ab (helices P2a and P2b with a 5/6-nt internal loop) and the minimal pseudoknot (P2b–P3 and associated loops). A helical extension of P2a, P2a.1, is specific to mammalian TR. Using NMR, we investigated the structures of the full-length TR pseudoknot and isolated subdomains in Oryzias latipes (Japanese medaka fish), which has the smallest vertebrate TR identified to date. We determined the solution NMR structure and studied the dynamics of medaka P2ab, and identified all base pairs and tertiary interactions in the minimal pseudoknot. Despite differences in length and sequence, the structure of medaka P2ab is more similar to human P2ab than predicted, and the medaka minimal pseudoknot has the same tertiary interactions as the human pseudoknot. Significantly, although P2a.1 is not predicted to form in teleost fish, we find that it forms in the full-length pseudoknot via an unexpected hairpin. Model structures of the subdomains are combined to generate a model of t/PK. These results provide evidence that the architecture for the vertebrate t/PK is conserved from teleost fish to human. The organization of the t/PK on telomerase reverse transcriptase for medaka and human is modeled based on the cryoEM structure of Tetrahymena telomerase, providing insight into function.

scholarly journals Identification of Kluyveromyces lactisTelomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain

1998 ◽  
Vol 18 (9) ◽  
pp. 4961-4970 ◽  
Author(s):  
Tracy Boswell Fulton ◽  
Elizabeth H. Blackburn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Telomerase Activity ◽  
Reaction Products ◽  
Telomeric Dna ◽  
Whole Cell ◽  
Cell Extracts ◽  
Dna Template ◽  
Insight Into

ABSTRACT Telomeres in the budding yeast Kluyveromyces lactisconsist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactistelomerase RNA, encoded by the TER1 gene, is ∼1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactiswhole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactistelomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactistelomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactistelomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.

111 CHARACTERIZATION OF BOVINE TELOMERASE REVERSE TRANSCRIPTASE DURING PREIMPLANTATION EMBRYOGENESIS

2008 ◽  
Vol 20 (1) ◽  
pp. 136
Author(s):  
P. Madan ◽  
W. A. King ◽  
D. H. Betts
Keyword(s):  
Reverse Transcriptase ◽  
Germinal Vesicle ◽  
Telomerase Activity ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Telomerase Reverse Transcriptase ◽  
Telomeric Dna ◽  
Cell Stage ◽  
Highly Active

Telomerase is a specialized reverse transcriptase that extends telomeric DNA of eukaryotic chromosomes and is composed of 2 essential subunits, the telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT). Together, this ribonucleoprotein complex is normally highly active in germ cells and stem/progenitor cells but not in normal somatic cells. In humans, this regulation of telomerase activity is primarily coordinated at the level of transcription of the TERT gene, whereas TERC is virtually ubiquitously expressed. Previous studies from our laboratory have detected telomerase activity in embryos from all stages of early bovine development. However, the regulation of the telomerase subunits remains poorly understood. Therefore, the objective of our study was to characterize the expression of bovine TERT (bTERT) during bovine preimplantation embryogenesis. UsingRT-PCR and immunofluorescence staining procedures (n = 20 embryos at timed stages of development; r = 3), we demonstrate that mRNA transcripts and protein for bTERT were detected in preimplantation bovine embryos from 1-cell to the blastocyst stage. The specificity of bTERT PCR products was confirmed by sequencing and demonstrated 94% sequence homology to the human hTERT cDNA sequence. In the immature oocyte, bTERT protein was localized within the germinal vesicle, and after 18–20 h of in vitro maturation, bTERT was observed as doublet foci co-localized with the condensed metaphase II meiotic stage chromosomes. Post-fertilization, the expression of bTERT was observed within the pronuclei. Through the initial cleavage stages, the expression of bTERT was variable, with some blastomeres showing punctuate staining and others exhibiting a uniform staining pattern. By the 8-16 cell stage, the embryos demonstrated a peri-nuclear presence of bTERT. However, in morula and blastocysts, bTERT was localized to the nuclei as demonstrated by co-localization with 42,6-diamidino-2-phenylindole staining. Based on this pattern of expression, it is tempting to speculate that bTERT could be playing an important role in regulating telomerase activity during early embryo development. The translocation of bTERT protein from the cytoplasm to the nucleus in morulae supports the telomere elongation event that is known to occur in mammalian embryos during the transition from the morula to blastocyst stages.

scholarly journals Processing of nontelomeric 3' ends by telomerase: default template alignment and endonucleolytic cleavage.

1996 ◽  
Vol 16 (7) ◽  
pp. 3437-3445 ◽  
Author(s):  
M Melek ◽  
E C Greene ◽  
D E Shippen
Keyword(s):  
De Novo ◽  
Telomeric Sequence ◽  
Telomeric Dna ◽  
Cleavage Reaction ◽  
Developmentally Regulated ◽  
Endonucleolytic Cleavage ◽  
Elongation Cycle ◽  
Dna Primers

Telomerase is a specialized reverse transcriptase that maintains telomeres at chromosome ends by extending preexisting tracts of telomeric DNA and forming telomeres de novo on broken chromosomes. Whereas the interaction of telomerase with telomeric DNA has been studied in some detail, relatively little is known about how this enzyme processes nontelomeric DNA. In this study we recruited the Euplotes telomerase to nontelomeric 3' termini in vitro using chimeric DNA primers that carried one repeat of a telomeric sequence at various positions upstream of a nontelomeric 3' end. Such primers were processed in two distinct pathways. First, nontelomeric 3' ends could be elongated directly by positioning a primer terminus at a specific site on the RNA template. Delivery to this default site was precise, always resulting in the addition of 4 dG residues to the non-telomeric 3' ends. These same residues initiate new telomeres formed in vivo. Alternatively, 3' nontelomeric nucleotides were removed from primers prior to initiating the first elongation cycle. As with default positioning of nontelomeric 3' ends, the cleavage event was extremely precise and was followed by the addition of dG residues to the primer 3' ends. The specificity of the cleavage reaction was mediated by primer interaction with the RNA template and, remarkably, proceeded by an endonucleolytic mechanism. These observations suggest a mechanism for the precision of developmentally regulated de novo telomere formation and expand our understanding of the enzymatic properties of telomerase.

scholarly journals Peculiarities of Yeasts and Human Telomerase RNAs Processing

Acta Naturae ◽  
2016 ◽  
Vol 8 (4) ◽  
pp. 14-22 ◽  
Author(s):  
M. P. Rubtsova ◽  
D. P. Vasilkova ◽  
Yu. V. Naraykina ◽  
O. A. Dontsova
Keyword(s):  
Reverse Transcriptase ◽  
Somatic Cells ◽  
Telomerase Activity ◽  
Proliferative Potential ◽  
Telomerase Rna ◽  
Embryonic Cells ◽  
Eukaryotic Chromosome ◽  
Human Telomerase ◽  
Linear Chromosomes

Telomerase is one of the major components of the telomeres -- linear eukaryotic chromosome ends - maintenance system. Linear chromosomes are shortened during each cell division due to the removal of the primer used for DNA replication. Special repeated telomere sequences at the very ends of linear chromosomes prevent the deletion of genome information caused by primer removal. Telomeres are shortened at each replication round until it becomes critically short and is no longer able to protect the chromosome in somatic cells. At this stage, a cell undergoes a crisis and usually dies. Rare cases result in telomerase activation, and the cell gains unlimited proliferative capacity. Special types of cells, such as stem, germ, embryonic cells and cells from tissues with a high proliferative potential, maintain their telomerase activity indefinitely. The telomerase is inactive in the majority of somatic cells. Telomerase activity in vitro requires two key components: telomerase reverse transcriptase and telomerase RNA. In cancer cells, telomerase reactivates due to the expression of the reverse transcriptase gene. Telomerase RNA expresses constitutively in the majority of human cells. This fact suggests that there are alternative functions to telomerase RNA that are unknown at the moment. In this manuscript, we review the biogenesis of yeasts and human telomerase RNAs thanks to breakthroughs achieved in research on telomerase RNA processing by different yeasts species and humans in the last several years.

scholarly journals Functional Multimerization of the Human Telomerase Reverse Transcriptase

2001 ◽  
Vol 21 (18) ◽  
pp. 6151-6160 ◽  
Author(s):  
Tara L. Beattie ◽  
Wen Zhou ◽  
Murray O. Robinson ◽  
Lea Harrington
Keyword(s):  
Reverse Transcriptase ◽  
Telomerase Activity ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Human Telomerase Reverse Transcriptase ◽  
Catalytic Core ◽  
Cell Extracts ◽  
Human Telomerase

ABSTRACT The telomerase enzyme exists as a large complex (∼1,000 kDa) in mammals and at minimum is composed of the telomerase RNA and the catalytic subunit telomerase reverse transcriptase (TERT). In Saccharomyces cerevisiae, telomerase appears to function as an interdependent dimer or multimer in vivo (J. Prescott and E. H. Blackburn, Genes Dev. 11:2790–2800, 1997). However, the requirements for multimerization are not known, and it remained unclear whether telomerase exists as a multimer in other organisms. We show here that human TERT (hTERT) forms a functional multimer in a rabbit reticulocyte lysate reconstitution assay and in human cell extracts. Two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity. This complementation requires the amino terminus of one hTERT and the reverse transcriptase and C-terminal domains of the second hTERT. The telomerase RNA must associate with only the latter hTERT for reconstitution of telomerase activity to occur. Multimerization of telomerase also facilitates the recognition and elongation of substrates in vitro and in vivo. These data suggest that the catalytic core of human telomerase may exist as a functionally cooperative dimer or multimer in vivo.

scholarly journals Two Inactive Fragments of the Integral RNA Cooperate To Assemble Active Telomerase with the Human Protein Catalytic Subunit (hTERT) In Vitro

1999 ◽  
Vol 19 (9) ◽  
pp. 6207-6216 ◽  
Author(s):  
Valerie M. Tesmer ◽  
Lance P. Ford ◽  
Shawn E. Holt ◽  
Bryan C. Frank ◽  
Xiaoming Yi ◽  
...  
Keyword(s):  
De Novo ◽  
Telomerase Activity ◽  
Catalytic Subunit ◽  
Human Protein ◽  
Cell Extracts ◽  
In Vitro Assembly ◽  
Reticulocyte Lysates ◽  
Minimal Sequence ◽  
Human Telomerase

ABSTRACT We have mapped the 5′ and 3′ boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3′ end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.

scholarly journals Localization of ferrochelatase in Plasmodium falciparum

2004 ◽  
Vol 384 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Sundaramurthy VARADHARAJAN ◽  
B. K. Chandrashekar SAGAR ◽  
Pundi N. RANGARAJAN ◽  
Govindarajan PADMANABAN
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Enzymic Activity ◽  
Malarial Parasite ◽  
Parasite Genome ◽  
Marker Proteins ◽  
Parasite Plasmodium ◽  
Theoretical Predictions ◽  
Parasite Plasmodium Falciparum

Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (δ-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.

Carcinogen-induced DNA amplification in vitro: overreplication of the simian virus 40 origin region in extracts from carcinogen-treated CO60 cells

1990 ◽  
Vol 10 (1) ◽  
pp. 75-83
Author(s):  
Y Berko-Flint ◽  
S Karby ◽  
D Hassin ◽  
S Lavi
Keyword(s):  
De Novo ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Dna Molecules ◽  
Viral Origin ◽  
Cell Extracts ◽  
Origin Region

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.

scholarly journals The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

2019 ◽  
Vol 116 (49) ◽  
pp. 24542-24550 ◽  
Author(s):  
Jiarui Song ◽  
Dhenugen Logeswaran ◽  
Claudia Castillo-González ◽  
Yang Li ◽  
Sreyashree Bose ◽  
...  
Keyword(s):  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Structural Element ◽  
Stem Loop ◽  
Evolutionary Pathway ◽  
Pol Iii

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

Sign in / Sign up

Export Citation Format

Share Document