Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have investigated three aspects of RNA turmor virus replication and cell transformation: (1) the properties of the purified avian and mammalian viral RNA-directed DNA polumerase, (2) some characteristics of the viral 60-70S RNA genome, 30-40S RNA subunits and intracellular viral RNA species, and (3) the interaction of the viral DNA polymerase with its RNA template early during infection and cell transformation by the murine sarcoma-leukemia virus (MSV[MLV]). Avian myeloblastosis virus (AMV) contains two forms of RNA-directed DNA polymerase, alpha, consisting of a single polypeptide of molecular weight 65,000, and alphabeta, consisting of two polypeptides of molecular weights 65,000 and 105,000. The alpha and alphabeta forms of AMV DNA polymerase both possess RNase H activity that requires free end termini on the ribopolymer and can degrade the RNA of the RNA-DNA hybrid in the 3' to 5' and 5' to 3' directions. But, alpha and alphabeta possess a different mode of exoribonuclease activity. While alphabeta RNase H is a processive exoribonuclease that degrades the polynucleotide chain to a core residue before attacking a second chain, alpha RNase H is a random exoribonuclease that releases the polynucleotide after each scission. Highly purified Moloney-MSV(MLV) DNA polymerase has both RNase H activity and the ability to read viral 60-70S RNA. These activities comigrate through five different steps of purification and are present at levels comparable to those found in purified AMV DNA polymerase. The MSV(MLV) 60-70S RNA genome and 35S RNA subunits were shown by periodate oxidationtritiated borohydride reduction to contain adenosine as the major 3'-terminal nucleoside. Poly (A) segments were isolated from viral 60-70S and 35S RNA by treatment with RNase A or RNase T1 and purified by afinity chromatography and gel electrophoresis. Viral poly(A) was shown to be present at the 3' terminus as -G(C,U)A190AOH. The similar sequence reported for poly(A) present in mammalian mRNA suggests that similar mechanisma are involved in the transcription and processing of both cellular and viral DNA sequences. Within transformed cells replicating MSV(MLV), viral 35S and 20S RNA were found in membrane-bound polyribosomes, whereas only 35S RNA was detected in free polyribosomes. The origin and function of 20S RNA is unknown. The early events during rapid infection and cell transformation of mouse 3T6 cells by the Harvey strain of MSV(MLV) were studied. By both autoradiographic analysis and molecular hybridization, viral DNA synthesis was detected in the cytoplasm by 1 hour after infection, reached a maximum at 2 hours, and subsequently decreased. Cytological chase experiments produced evidence that cytoplasmic viral DNA was transported to the nucleus. In situ hybridization experiments using radioactive viral DNA product as a probe demonstrated the rapid association of viral DNA sequences with the chromocenters of interphase nuclei and with the centromeric heterochromatin regions of some chromosomes.
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PMID:Properties of oncornavirus RNA-directed DNA polymerase, the RNA template, and the intracellular products formed early during infection and cell transformation. 5 Sep 2

The RNA moiety of the ribonucleoprotein enzyme telomerase from the ciliate Euplotes crassus was identified and its gene was sequenced. Functional analysis, in which oligonucleotides complementary to portions of the telomerase RNA were tested for their ability to prime telomerase in vitro, showed that the sequence 5' CAAAACCCCAAA 3' in this RNA is the template for synthesis of telomeric TTTTGGGG repeats by the Euplotes telomerase. The data provide a direct demonstration of a template function for a telomerase RNA and demarcate the outer boundaries of the telomeric template. Telomerase can now be defined as a specialized reverse transcriptase.
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PMID:Functional evidence for an RNA template in telomerase. 168 74

Cytological preparations of interphase nuclei and chromosomes from mouse 3T6 cells prepared at various times after infection with the murine sarcomaleukemia virus complex were hybridized with the [(3)H]DNA product of the viral RNA-directed DNA polymerase. While uninfected nuclei had an average of 4 autoradiographic grains, infected nuclei had 30 grains at 5 hr after infection and 63-65 grains at 11 and 25 hr. Virus-specific grains were localized in the chromocenters of interphase nuclei and were found also in the centromeric heterochromatin region of metaphase chromosomes. These findings provide evidence that the viral RNA-directed DNA polymerase functions to synthesize virus-specific DNA early after infection and that newly synthesized viral DNA rapidly becomes associated with or integrated into specific intranuclear sites.
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PMID:Detection and localization of virus-specific DNA by in situ hybridization of cells during infection and rapid transformation by the murine sarcoma-leukemia virus. 413 10

Macronuclear telomeres in Oxytricha exist as DNA-protein complexes in which the termini of the G-rich strands are bound by a 97-kDa telomere protein. During telomeric DNA replication, the replication machinery must have access to the G-rich strand. However, given the stability of telomere protein binding, it has been unclear how this is accomplished. In this study we investigated the ability of several different DNA polymerases to access telomeric DNA in Oxytricha telomere protein-DNA complexes. Although DNA bound by the telomere protein is not degraded by micrococcal nuclease or labeled by terminal deoxynucleotidyltransferase, this DNA serves as an efficient primer for the addition of telomeric repeats by telomerase, a specialized RNA-dependent DNA polymerase (ribonucleoprotein reverse transcriptase), EC 2.7.7.49. Moreover, in the presence of a suitable complementary C-rich DNA template, AMV reverse transcriptase and the E. coli Klenow fragment will also elongate DNA bound by the telomere protein. These findings indicate that the 3' terminus and the Watson-Crick base pairing positions are exposed in the protein complex. We propose that the telomere protein can serve a dual role at the telomere by protecting the DNA phosphate backbone from degradation while simultaneously exposing the DNA bases for replication.
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PMID:DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases. 750 21

We characterized TRAS1, a retrotransposable element which was inserted into the telomeric repetitive sequence (CCTAA)n of the silkworm, Bombyx mori. The complete sequence of TRAS1, a stretch of 7.8 kb with a poly(A) tract at the 3' end, was determined. No long terminal repeat (LTR) was found at the termini of the element. TRAS1 contains gag- and pol-like open reading frames (ORFs) which are similar to those of non-LTR retrotransposons. The two ORFs overlap but are one nucleotide out of frame (+1 frameshift). Most of the approximately 250 copies of TRAS1 elements in the genome were highly conserved in the structure. Chromosomal in situ hybridization showed that TRAS1 elements are clustered at the telomeres of Bombyx chromosomes. A phylogenetic analysis using the amino acid sequence of the reverse transcriptase domain within the pol-like ORF revealed that TRAS1 falls into one lineage with R1, which is a family of non-LTR retrotransposons inserted into the same site within the 28S ribosomal DNA unit in most insects. TRAS1 may have been derived from R1 and changed the target specificity so that TRAS1 inserts into the telomeric repetitive sequence (CCTAA)n. Southern hybridization and Bal 31 exonuclease analyses showed that TRAS1 elements are clustered proximal to the terminal long tract of (CCTAA)n. TRAS1 is a novel family of non-LTR retrotransposons which are inserted into the telomeric repetitive sequences as target sites.
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PMID:Structural analysis of TRAS1, a novel family of telomeric repeat-associated retrotransposons in the silkworm, Bombyx mori. 762 45

Telomerase, an essential ribonucleoprotein reverse transcriptase, adds telomeric DNA to the ends of eukaryotic chromosomes. We examined the conformational properties of the naked RNA moiety of telomerase from two related ciliates, Tetrahymena thermophila and Glaucoma chattoni. As well as finding evidence for features proposed previously on the basis of phylogenetic comparisons, novel conserved structural properties were revealed. Specifically, although the region around helix III was previously proposed to form a pseudoknot, our results indicate that in the naked RNA this region maintains a level of 'plasticity', probably in an equilibrium favoring one of two helices. In addition, these studies reveal that the templating domain is not entirely single-stranded as previously proposed, but is ordered due to constraints imposed by other parts of the RNA. Finally, our results suggest that the GA bulge in helix IV may introduce a structurally conserved kink. We now propose a 'two-domain' structure for the telomerase RNA based on function: one conformationally flexible domain, which includes the template and the region around helix III, involved with enzymatic function, and a second largely helical domain, including helices I and IV and the proposed kink, which may serve as a scaffold for protein binding.
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PMID:Architecture of telomerase RNA. 798 68

The ribonucleoprotein enzyme telomerase is a specialized type of cellular reverse transcriptase which synthesizes one strand of telomeric DNA, using as the template a sequence in the RNA moiety of telomerase. We analyzed the effects of various nucleoside analogs, known to be chain-terminating inhibitors of retroviral reverse transcriptases, on Tetrahymena thermophila telomerase activity in vitro. We also analyzed the effects of such analogs on telomere length and maintenance in vivo, and on vegetative growth and mating of Tetrahymena cells. Arabinofuranyl-guanosine triphosphate (Ara-GTP) and ddGTP both efficiently inhibited telomerase activity in vitro, while azidothymidine triphosphate (AZT-TP), dideoxyinosine triphosphate (ddITP) or ddTTP were less efficient inhibitors. All of these nucleoside triphosphate analogs, however, produced analog-specific alterations of the normal banding patterns seen upon gel electrophoresis of the synthesis products of telomerase, suggesting that their chain terminating and/or competitive actions differ at different positions along the RNA template. The analogs AZT, 3'-deoxy-2',3'-didehydrothymidine (d4T) and Ara-G in nucleoside form caused consistent and rapid telomere shortening in vegetatively growing Tetrahymena. In contrast, ddG or ddI had no effect on telomere length or cell growth rates. AZT caused growth rates and viability to decrease in a fraction of cells, while Ara-G had no such effects even after several weeks in culture. Neither AZT, Ara-G, acycloguanosine (Acyclo-G), ddG nor ddI had any detectable effect on cell mating, as assayed by quantitation of the efficiency of formation of progeny from mated cells. However, AZT decreased the efficiency of programmed de novo telomere addition during macronuclear development in mating cells.
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PMID:The effects of nucleoside analogs on telomerase and telomeres in Tetrahymena. 815 19

We have designed a single polymerase chain reaction (PCR) primer pair that detects the MLL/AF-4 fusion mRNA encoded by the derivative 11 chromosome from t(4;11)(q21;q23) leukemia cells using the reverse transcriptase PCR technique. PCR amplification was possible in seven of seven cells studied. Sequencing of the amplified products showed three different breakpoints on 11q23 and three on 4q21, resulting in six unique fusion sequences. All fusion sequences maintained an open reading frame. The areas of the MLL and AF-4 genes that are conserved in all derivative 11 fusion RNAs and therefore likely to contribute to the function of the oncogenic fusion protein are centromeric regions of MLL through exon 6 (retaining the AT hook motif) and telomeric regions of AF-4 beginning at codon 491 (containing nuclear localization and GTP-binding motifs). A single primer pair was able to detect the derivative 11 fusion transcript in seven of seven cases of t(4;11) acute leukemia tested. Given the variability shown in specific fusion sequences, studies correlating differential exon usage with clinical parameters will require different fusion-specific oligonucleotides or PCR primer pairs.
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PMID:Heterogeneity in MLL/AF-4 fusion messenger RNA detected by the polymerase chain reaction in t(4;11) acute leukemia. 835 9

The ribonucleoprotein telomerase, a specialized cellular reverse transcriptase, synthesizes one strand of the telomeric DNA of eukaryotes. We analyzed telomere maintenance in two immortalized human cell lines: the B-cell line JY616 and the T-cell line Jurkat E6-1, and determined whether known inhibitors of retroviral reverse transcriptases could perturb telomere lengths and growth rates of these cells in culture. Dideoxyguanosine (ddG) caused reproducible, progressive telomere shortening over several weeks of passaging, after which the telomeres stabilized and remained short. However, the prolonged passaging in ddG caused no observable effects on cell population doubling rates or morphology. Azidothymidine (AZT) caused progressive telomere shortening in some but not all T- and B-cell cultures. Telomerase activity was present in both cell lines and was inhibited in vitro by ddGTP and AZT triphosphate. Prolonged passaging in arabinofuranyl-guanosine, dideoxyinosine (ddI), dideoxyadenosine (ddA), didehydrothymidine (d4T), or phosphonoformic acid (foscarnet) did not cause reproducible telomere shortening or decreased cell growth rates or viabilities. Combining AZT, foscarnet, and/or arabinofuranyl-guanosine with ddG did not significantly augment the effects of ddG alone. Strikingly, with or without inhibitors, telomere lengths were often highly unstable in both cell lines and varied between parallel cell cultures. We propose that telomere lengths in these T- and B-cell lines are determined by both telomerase and telomerase-independent mechanisms.
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PMID:Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines. 852 29

The ribonucleoprotein enzyme telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA. Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization. Position 49 of the telomerase RNA defined the 3' templating residue boundary, demonstrating that seven positions, residues 43 to 49, are capable of acting as templating residues. We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with telomerase RNA residues. Unexpectedly, formation of a Watson-Crick base pair specifically between the primer DNA and telomerase RNA residue 50 is critical in promoting primer elongation. In contrast, mutant telomerase with the cytosine at position 49 mutated to a G exhibited efficient 3' mispair extension. This work provides new evidence for specific primer-telomerase interactions, as well as base-specific interactions involving the telomerase RNA, playing roles in essential active-site functions of telomerase.
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PMID:Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase. 852 30


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