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)

Phytohaemagglutinin stimulates lymphoid cells to initiate active cell division which is tightly coordinated with transcription of ribosomal RNA genes. Nuclear Run-On assays demonstrated that treatment of peripheral blood lymphocytes with PHA (10 microg/ml) resulted in maximal rRNA synthesis after 64 hrs. In contrast, mRNA levels for upstream binding factor (UBF)1 and UBF2, as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, increased relatively rapidly within 3 to 6 hrs. and remained elevated for at least the next 60 hrs. We further showed that exponentially growing cells of promyelocytic leukemia line HL-60 contained the same amounts of UBF1 and UBF2 mRNAs as phytohaemagglutinin (PHA)-stimulated lymphocytes for 6 hrs. Growth arrest of HL-60 cells, caused by 10 nM phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic/macrophage-like differentiation for 72 hrs., has been accompanied by a 50% decrease in UBF1/2 mRNAs expression. The lowest concentrations of UBF1/2 mRNAs were revealed in non-dividing terminally differentiated granulocytes. Regardless the activity of RNA polymerase I transcription and cell division rate, UBF1 mRNA levels prevailed over UBF2 mRNA levels in all human blood cell populations tested. Our results suggest that UBF gene expression is an important regulatory mechanism involved in the acceleration and possibly deceleration of rDNA transcription observed during mitogenic stimulation and inhibition of blood cells.
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PMID:Early gene expression of both RNA polymerase I transcription factors UBF1 and UBF2 precedes ribosomal RNA synthesis during lymphocyte mitogenic stimulation. 959 85

Retroviruses require a specific host cellular tRNA primer for initiation of first-strand DNA synthesis. This primer is bound by viral proteins and copackaged into virions. We have exploited this property in the design and testing of an antiviral ribozyme fused to tRNA(3Lys), the primer used for lentiviral replication, including human immunodeficiency virus (HIV-1 and HIV-2). The chimera consists of tRNA(3Lys) covalently attached to a hammerhead ribozyme, which is targeted to the region immediately upstream of the primer binding site of the HIV-1 genome. The tRNA-ribozyme chimeric transcript is catalytically active in vitro and is efficiently bound by HIV reverse transcriptase with an affinity similar to that of tRNA(3Lys). We have expressed the chimeric RNAs from either the tRNA(3Lys) intragenic RNA polymerase III promoter or from a human U6 snRNA promoter. The U6 promoter results in up to 10-fold enhanced expression of the tRNA-ribozyme. Most importantly, the tRNA(3Lys)-ribozymes are encapsidated in HIV-1 virions such that they are effective in substantially reducing the level of infectious virus produced from cells cotransfected with HIV-1 proviral DNA. These results demonstrate the feasibility of using this novel strategy to reduce HIV infectivity and more generally indicate the potential power of using the retroviral primer tRNAs as tools for expressing and delivering ribozymes and other antiretroviral RNAs to the virion capsid.
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PMID:Virion encapsidation of tRNA(3Lys)-ribozyme chimeric RNAs inhibits HIV infection. 966 56

Two catalytic functions were required, minimally, for the appearance of DNA in evolution: a ribonucleotide reductase (RNR) and a reverse transcriptase (RT). If one accepts the explanatory strength of the RNA world model, it is clear that DNA molecules arose in the RNA world at some stage during the early evolution of cells. I suggest that competition for limited and valuable resources such as nucleotides, amino acids, and sugars made an early appearance among RNA cells, RNA viruses, viroids, and RNA plasmids. Structural and functional similarities between the different types of polymerases favor the simple hypothesis that the first RTs were RNA polymerase mutants that preferentially joined together preexisting deoxyribonucleotide triphosphates (dNTPs) using RNA templates. What was the role of dNTPs inside cells before DNA was synthesized and tested by natural selection? The oxygen atom that is removed by the reductase is of crucial importance to many ribozyme functions, since the 2'-OH is a strong nucleophile that forms transitional states during catalysis. Consequently, a RNR may have been used by cellular parasites to inhibit ribozyme action. Thus, DNA may have been, initially, an inert by-product of retrotranscription in lineages that acquired RTs and could synthesize DNA molecules using cellular RNA templates to detoxify the intracellular environment. DNA was useless as template until a transcriptase (DNA-dependent RNA polymerase) evolved that could copy (-)DNA to reconstitute the (+)RNA genome, indeed a successful way of confronting ribonuclease threats in the RNA world.
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PMID:Inhibition of ribozymes by deoxyribonucleotides and the origin of DNA. 969 60

Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3' ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3' ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.
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PMID:Mutations in nonconserved domains of Ty3 integrase affect multiple stages of the Ty3 life cycle. 984 51

Simple sequence repeat telomeric DNA is maintained by a specialized reverse transcriptase, telomerase. The integral RNA subunit of telomerase contains a template region that determines the sequence added to chromosome ends. Aside from providing the template, little is known about the role of the telomerase RNA. In addition, no hypotheses have been suggested to account for the striking evolutionary divergence in size and sequence between telomerase RNAs of ciliates, yeasts, and mammals. We show that the two- to threefold increase in size of the mammalian telomerase RNAs relative to ciliate telomerase RNAs is due to the presence of an extra domain resembling a box H/ACA small nucleolar RNA (snoRNA). The human telomerase RNA (hTR) H/ACA domain is essential in vivo for hTR accumulation, hTR 3' end processing, and telomerase activity. By substituting the U64 box H/ACA snoRNA for the hTR H/ACA domain, we demonstrate that a heterologous snoRNA can function to promote chimeric RNA accumulation and 3' end processing but not telomerase activity. In addition, we show that maturation of full-length hTR and its assembly into active telomerase occur from an mRNA promoter-driven RNA polymerase II transcript but not from a U6 snRNA promoter-driven RNA polymerase III transcript. Finally, we show that a small percentage of hTR is associated with nucleoli. These results have implications for the biogenesis and structure of hTR and the human telomerase ribonucleoprotein complex. They also expand the structural and functional diversity of the box H/ACA snoRNA motif.
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PMID:A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3' end. 985 80

Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes and maintains telomeric DNA. Studies of telomeres and telomerase are facilitated by the large number of linear DNA molecules found in ciliated protozoa, such as Tetrahymena thermophila. To examine the expression of telomerase, we investigated the transcription of the RNA polymerase III-directed gene encoding the RNA subunit (TER1) of this enzyme. A chimeric gene containing the Glaucoma chattoni TER1 transcribed region flanked by 5' and 3' Tetrahymena regions was used to identify promoter elements following transformation of Tetrahymena cells. Disruption of a conserved proximal sequence element (PSE) located at -55 in the Tetrahymena TER1 5' flanking region eliminated expression of the chimeric gene. In addition, mutation of an A/T-rich element at -25 decreased expression markedly. A gel mobility shift assay and protein-DNA cross-linking identified a PSE-binding polypeptide of 50-60 kDa in Tetrahymena extracts. Gel filtration analysis revealed a native molecular mass of approximately 160 kDa for this binding activity. Our results point to a similar architecture between ciliate telomerase RNA and metazoan U6 small nuclear RNA promoters.
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PMID:Identification of an essential proximal sequence element in the promoter of the telomerase RNA gene of Tetrahymena thermophila. 1051 20

Repetitive elements flanked by exons 2 and 3 of the human transaldolase gene, thus termed transaldolase-associated repetitive elements, TARE, were identified in human DNA. Nonpolyadenylated TARE transcripts were detected by Northern blot analysis and cloned by reverse transcriptase-mediated polymerase chain reaction from human T lymphocytes. A dominant 1085-nucleotide long transcript, TARE-6, contained two adjacent Alu elements, a right monomer and a complete dimer, oriented opposite to the direction of transcription of the transaldolase gene. Reverse transcriptase-polymerase chain reaction and in vitro transcription analyses showed that transcription of TARE-6 proceeded in the orientation of the RNA pol III promoter of the Alu dimer and opposite to the orientation of the TAL-H gene. TAREs lacking RNA polymerase III promoter showed no transcriptional activity. In vitro transcription of TARE-6 was resistant to 1 microg/ml alpha-amanitin but sensitive to 100 microg/ml alpha-amanitin and tagetitoxin, suggesting involvement of RNA polymerase III. TAREs in both the transaldolase and HSAG-1 genomic loci were surrounded by TA target site duplications. Homologies between transaldolase and HSAG-1 break off internally at splice donor and acceptor sites. The results suggest RNA polymerase III-mediated transcription of TARE may be a source of repetitive elements, contributing to distinct genes and thus shaping the human genome.
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PMID:Human transaldolase-associated repetitive elements are transcribed by RNA polymerase III. 1070 96

In this study we have designed and constructed an anti-Fas ribozyme and show that it can specifically cleave the Fas mRNA in vitro. Moreover, to test its efficacy ex vivo, we transfected the anti-Fas ribozyme into betaTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expression. Like pancreatic beta cells, betaTC-3 cells do not constitutively express Fas, but Fas expression can be induced with IL-1 and IFN-gamma. Transfected cells expressed an average of 5000 copies of anti-Fas ribozyme transcript per cell as assessed by reverse transcriptase-real-time PCR. After IL-1/IFN-gamma treatment, betaTC-3 cells transfected with the anti-Fas ribozyme expressed 80% less Fas compared with mock-transfected cells. In addition, the anti-Fas ribozyme also inhibited Fas expression in NIT-1 insulinoma cells and in primary cultures of dispersed pancreatic islet cells. Inhibition of de novo Fas expression in betaTC-3 cells expressing the anti-Fas ribozyme correlated with resistance to Fas-mediated apoptosis as determined by the number of cells exhibiting caspase 3 proteolytic activity. Hence, we have engineered a ribozyme capable of preventing Fas expression in the betaTC-3 pancreatic insulinoma cell line and conferring resistance to Fas-mediated apoptosis. We suggest that ribozymes may be potentially useful to engineer resistance to apoptosis in transplantable beta cells, a feature that may significantly improve the survival of islet cell grafts.
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PMID:Inhibition of Fas-mediated apoptosis in mouse insulinoma betaTC-3 cells via an anti-Fas ribozyme. 1081 Dec 32

Short interspersed elements (SINEs), typified by the human Alu repeat, are RNA polymerase III (pol III)-transcribed sequences that replicate within the genome through an RNA intermediate. Replication of SINEs has been extensive in mammalian evolution: an estimated 5% of the human genome consists of Alu repeats. The mechanisms regulating transcription, reverse transcription, and reinsertion of SINE elements in genomic DNA are poorly understood. Here we report that expression of murine SINE transcripts of both the B1 and B2 classes is strongly upregulated after prolonged exposure to cisplatin, etoposide, or gamma radiation. A similar induction of Alu transcripts in human cells occurs under these conditions. This induction is not due to a general upregulation of pol III activity in either species. Genotoxic treatment of murine cells containing an exogenous human Alu element induced Alu transcription. Concomitant with the increased expression of SINEs, an increase in cellular reverse transcriptase was observed after exposure to these same DNA-damaging agents. These findings suggest that genomic damage may be an important activator of SINEs, and that SINE mobility may contribute to secondary malignancy after exposure to DNA-damaging chemotherapy.
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PMID:Transcriptional activation of short interspersed elements by DNA-damaging agents. 1110 77

Influenza B virus causes a significant amount of morbidity and mortality, yet the systems to produce high yield inactivated vaccines for these viruses have lagged behind the development of those for influenza A virus. We have established a plasmid-only reverse genetics system for the generation of recombinant influenza B virus that facilitates the generation of vaccine viruses without the need for time consuming coinfection and selection procedures currently required to produce reassortants. We cloned the eight viral cDNAs of influenza B/Yamanashi/166/98, which yields relatively high titers in embryonated chicken eggs, between RNA polymerase I and RNA polymerase II transcription units. Virus was detected as early as 3 days after transfection of cocultured COS7 and Madin-Darby canine kidney cells and achieved levels of 10(6)-10(7) plaque-forming units per ml of cell supernatant 6 days after transfection. The full-length sequence of the recombinant virus after passage into embryonated chicken eggs was identical to that of the input plasmids. To improve the utility of the eight-plasmid system for generating 6 + 2 reassortants from recently circulating influenza B strains, we optimized the reverse transcriptase-PCR for cloning of the hemagglutinin (HA) and neuraminidase (NA) segments. The six internal genes of B/Yamanashi/166/98 were used as the backbone to generate 6 + 2 reassortants including the HA and NA gene segments from B/Victoria/504/2000, B/Hong Kong/330/2001, and B/Hawaii/10/2001. Our results demonstrate that the eight-plasmid system can be used for the generation of high yields of influenza B virus vaccines expressing current HA and NA glycoproteins from either of the two lineages of influenza B virus.
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PMID:Rescue of influenza B virus from eight plasmids. 1217 12


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