Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of the purified poliovirus RNA-dependent RNA polymerase to viral and nonviral RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. Optimal conditions for the binding of purified polymerase (fraction 5-PAS) to 32P-labeled poliovirion RNA were determined. The binding of purified polymerase to 32P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) much much greater than poly(U) greater than poly(C) greater than poly(A). In competitive binding studies, the polymerase bound with equal efficiency to virion RNA and to a subgenomic transcript which contained the 3' end of the genome. The polymerase bound to 18S ribosomal RNA and to globin mRNA equally well, but with a five-fold lower affinity than to virus-specific RNAs. The results suggest that the polymerase exhibits sequence specificity in binding and that polymerase binding sites in poliovirus RNA may contain (G- and/or U)-rich sequences.
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PMID:Measurement of poliovirus RNA polymerase binding to poliovirion and nonviral RNAs using a filter-binding assay. 246 45

The multiplication of murine coronavirus strains A59 or JHM in Sac(-) cells was unaffected by the presence of alpha-amanitin at concentrations which inhibited the host cell DNA-dependent RNA polymerase activity. In cells infected with the A59 virus strain, actinomycin D-resistant RNA synthesis could readily be detected by pulse-labelling with [3H]uridine; this virus-specific RNA synthesis was not induced in the presence of the protein synthesis inhibitor anisomycin. A new RNA-dependent RNA polymerase activity was detected in the large particle fraction of A59 virus-infected cells. Optimal conditions for enzyme activity in vitro were established. Maximum activity occurred 5 h after infection, coincident with the peak of virus-specific RNA synthesis detected by pulse-labelling in vivo.
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PMID:RNA-dependent RNA polymerase activity in murine coronavirus-infected cells. 629 95

Three double-stranded RNA segments of bacteriophage phi 6 (L, M, and S) were transcribed in vitro by a virion-associated RNA polymerase. Regulation of L transcription was distinct from regulation of M and S transcription. Transcription of the L segment, which codes for early proteins, required manganous ion and high concentrations of all four ribonucleoside triphosphates and was inhibited by polyamines such as spermine. Transcription of the M and S segments, which code for late proteins, required manganous or magnesium ion and relatively low concentrations of all ribonucleoside triphosphates except GTP and was enhanced by polyamines. Optimal conditions for L transcription were more stringent than those for M and S transcription. These two apparently different patterns produced in in vitro transcription presumably reflect the two distinct in vivo transcription patterns; i.e., (i) similar amounts of three single-stranded RNA species were transcribed from the three corresponding segments of double-stranded RNA (early pattern) and (ii) a much larger amount of single-stranded RNA species was transcribed from M and S segments than from the L segment (late pattern). The early transcription pattern may be changed into the late pattern by a change of environment, such as substrate concentration. This suggests that the different enzymatic properties under the different environmental conditions of the virion-associated transcriptase are responsible for the transcriptional regulation throughout the infection cycle of bacteriophage phi 6.
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PMID:Transcriptional regulation of three double-stranded RNA segments of bacteriophage phi 6 in vitro. 682 50

This study describes the distinct roles of B7 and LFA-3 in the regulation of T cell responses. Activation of CD4+ T cells with Chinese hamster ovary (CHO)-DR4/B7 and CHO-DR4/LFA-3 cells that present the superantigen staphylococcal enterotoxin A resulted in significant T cell proliferation and substantial production of TNF and IFN-gamma. Strong IL-2 production was recorded in B7-costimulated, but not LFA-3-costimulated, cultures. The presence of B7 induced a more vigorous and prolonged proliferative T cell response compared with LFA-3 costimulation. In contrast, LFA-3 was more efficient than B7 in mediating cell adhesion of CD4+ T cells. Costimulation with the CHO-DR4/B7/LFA-3 triple transfectant resulted in enhanced cell adhesion, proliferation, and cytokine production compared with either DR4/B7 or DR4/LFA-3 alone. Optimal production of IL-2 by naive and memory CD4+ T cells was seen only when cells were costimulated with B7, whereas IFN-gamma production was induced in memory cells by both LFA-3 and B7. The Jurkat T cell line responded to CHO-DR4/B7/LFA-3 in a manner similar to peripheral blood CD4+ T cells. Reverse transcriptase-PCR analysis of Jurkat cells stimulated with staphylococcal enterotoxin E and the different CHO transfectants revealed that the cooperative effect of B7 and LFA-3 on IL-2 production was also seen at the mRNA level. The large amounts of IL-2 produced by B7 costimulation indicate a paracrine function of the B7/CD28 pathway, whereas the LFA-3/CD2 pathway provides strong adhesion and may facilitate autocrine T cell expansion. Combined expression of the B7 and LFA-3 molecules seems to provide an optimal Ag-presenting function that ensures strong adhesion and optimal signal transduction.
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PMID:Costimulation of human CD4+ T lymphocytes with B7 and lymphocyte function-associated antigen-3 results in distinct cell activation profiles. 752 63

Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.
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PMID:Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia. 765 5

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is considered to possess RNA-dependent RNA polymerase (RdRp) activity and to play an essential role for the viral replication. In this study, we expressed the NS5B protein of 65 kd by a recombinant baculovirus. With the highly purified NS5B protein, we established an in vitro system for RdRp activity by using poly(A) as a template and a 15-mer oligo(U) (oligo(U)15) as a primer. Optimal conditions of temperature and pH for primer-dependent polymerase activity of the NS5B were 32 degrees C and pH 8.0. The addition of 10 mmol of Mg2+ increased the activity. The importance of three motifs conserved in RdRp among other positive-strand RNA viruses was confirmed by introduction of an Ala residue to every amino acid of the motifs by site-directed mutagenesis. All mutants lost RdRp activity, but retained the RNA binding activity, except one mutant at Thr287/Asn291. Deletion mutant analysis indicated that the N-terminal region of NS5B protein was critical for the RNA binding. Inhibition of RdRp activity by (-)beta-L-2', 3'-dideoxy-3'-thiacytidine 5'-triphosphate (3TC; lamivudine triphosphate) and phosphonoacetic acid (PAA) was observed after screening of nucleoside analogs and known polymerase inhibitors. These data provide us not only important clues for understanding the mechanism of HCV replication, but also a new target of antiviral therapy.
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PMID:Expression of hepatitis C virus NS5B protein: characterization of its RNA polymerase activity and RNA binding. 1009 69

Previous studies have demonstrated that the c-kit encoded tyrosine kinase receptor and its ligand, steel factor (SLF), are critical for normal blood cell development. We have reported that transduction of the c-kit gene into single hematopoietic progenitor cells (HPC), CD34(+++) cells, from cord blood (CB) enhances erythroid colony formation via a SLF-dependent mechanism. We therefore decided to evaluate the impact on cell proliferation of co-transducing c-kit and SLF cDNAs into these cells. CD34(+++) cells were sorted as a population or as 1 cell/well for cells expressing the highest levels of CD34 and different levels of c-kit. Cells were then prestimulated with granulocyte macrophage (GM)-colony stimulating factor (CSF), interleukin (IL)-3, IL-6, erythropoietin (Epo) in the presence and absence of various concentrations of SLF. Cells were then transduced with SLF and/or c-kit cDNAs, and then assayed for colony formation with the same cytokine combination. At a single cell level, co-transduction with c-kit and SLF genes significantly enhanced colony formation compared with individual gene transduction, especially by erythroid and multipotential progenitors that responded to stimulation by added cytokines. Little or no growth was seen with the c-kit- and/or SLF-transduced cells without addition of cytokines. The degree of enhancement effected by co-transduction inversely correlated with the degree of expression of c-kit protein before transduction. Optimal enhancing effects were noted in CD34(+++) kit(Lo/-) cells co-transduced with both c-kit and SLF cDNAs. Reverse transcriptase-polymerase chain (RT-PCR) analysis of SLF mRNA expression in CD34(+++) cells and enzyme-linked immunoadsorbent assay (ELISA) measurement of secreted SLF protein demonstrated that the transduced SLF cDNA was expressed and soluble SLF was released in medium cultured with SLF gene transduced MACS-separated CD34(+) cells in the presence, but not in the absence, of IL-3, GM-CSF, IL-6, and Epo. These results demonstrate the enhancement of the proliferation of growth factor responsive HPC that express transduced c-kit and SLF genes.
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PMID:Co-transduction of cDNAs for c-kit and steel factor into single CD34+ cord blood cells further enhances the growth of erythroid and multipotential progenitors. 1117 93

The HeLa cell terminal uridylyltransferase (TUTase) that specifically modifies the 3'-end of mammalian U6 small nuclear RNA (snRNA) was characterized with respect to ionic dependence and substrate requirements. Optimal enzyme activity was obtained at moderate ionic strength (60 mm KCl) and depended on the presence of 5 mm MgCl2. In vitro synthesized U6 snRNA without a 3'-terminal UMP residue was not accepted as substrate. In contrast, U6 snRNA molecules containing one, two or three 3'-terminal UMP residues were filled up efficiently, generating the 3'-terminal structure with four UMP residues observed in newly transcribed cellular U6 snRNA. In this reaction, the addition of more than one UMP nucleotide depended on higher UTP concentrations. The analysis of internally mutated U6 snRNA revealed that the fill-in reaction by the U6-TUTase was not controlled by opposite-strand nucleotides, excluding an RNA-dependent RNA polymerase mechanism. Furthermore, electrophoretic mobility-shift analyses showed that the U6-TUTase was able to form stable complexes with the U6 snRNA in vitro. On the basis of these findings, a protocol was developed for affinity purification of the enzyme. In agreement with indirect labeling results, PAGE of a largely purified enzyme revealed an apparent molecular mass of 115 kDa for the U6-TUTase.
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PMID:Biochemical characterization of a U6 small nuclear RNA-specific terminal uridylyltransferase. 1260 30

Replication of hepatitis C virus (HCV) RNA is catalyzed by the virally encoded RNA-dependent RNA polymerase NS5B. It is believed that the viral polymerase utilizes a de novo or primer-independent mechanism for initiation of RNA synthesis. Our previous work has shown that dinucleotides were efficient initiation molecules for NS5B in vitro (W. Zhong, E. Ferrari, C. A. Lesburg, D. Maag, S. K. Ghosh, C. E. Cameron, J. Y. Lau, and Z. Hong, J. Virol. 74:9134-9143, 2000). In this study, we further demonstrated that dinucleotide analogues could serve as inhibitors of de novo initiation of RNA synthesis directed by HCV NS5B. Both mononucleotide- and dinucleotide-initiated RNA syntheses were affected by dinucleotide analogues. The presence of the 5'-phosphate group in the dinucleotide compounds was required for efficient inhibition of de novo initiation. Optimal inhibitory activity also appeared to be dependent on the base-pairing potential between the compounds and the template terminal bases. Because the initiation process is a rate-limiting step in viral RNA replication, inhibitors that interfere with the initiation process will have advantages in suppressing virus replication. The use of dinucleotide analogues as inhibitor molecules to target viral replication initiation represents a novel approach to antiviral interference.
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PMID:Dinucleotide analogues as novel inhibitors of RNA-dependent RNA polymerase of hepatitis C Virus. 1287 40

Uncoupling proteins (UCPs) present in the inner mitochondrial membrane are involved in uncoupling respiration from ATP synthesis. Five UCP isoforms have been identified but information about their presence and level of expression in the central nervous system remains incomplete. To determine the nature and proportion of UCP isoform mRNAs present in brain cortex, we developed and optimized a specific quantitative reverse-transcription polymerase chain reaction procedure. Optimal range of RNA concentrations to be used in the reverse-transcriptase reaction was determined. Primer design and concentration were optimized for each target gene while polymerase chain reaction efficiency was assessed for a range of reverse-transcriptase dilutions. Genomic contribution to the quantitative signal was evaluated for each isoform and minimized. Three reference genes were tested for normalization, and beta-actin was found to be the most stable among tissues. Results indicate that brain cortex contains significant amounts of all UCP mRNAs, with UCP5 and UCP4 being the most abundant, as opposed to brown adipose tissue and skeletal muscle, which predominantly express UCP1 and UCP3, respectively. These data provide a first quantitative assessment of UCP mRNA expression in mouse brain, showing the presence of all five isoforms with distinct proportions, thus suggesting specific roles in the central nervous system.
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PMID:Quantitative rt-PCR analysis of uncoupling protein isoforms in mouse brain cortex: methodological optimization and comparison of expression with brown adipose tissue and skeletal muscle. 1524 Nov 86


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