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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A fully automated instrument for multiple simultaneous peptide synthesis was constructed to provide large numbers of peptides for immunological research. The synthesis is performed in a flow-through mode with the conventional solid supports contained in 48 individual reaction columns. The instrument is based on a commercial autosampler equipped with a motor-driven syringe for accurate delivery of reagents and a robot arm carrying a dispenser needle. Dedicated software was developed to compile overlapping peptides from a given protein sequence and to control all functions of the robot. In situ activation by BOP was chosen as the optimized chemistry protocol. The peptides are cleaved from the resin in the reactors used for synthesis, thus minimizing handling. Performance of the instrument was demonstrated by synthesis of overlapping 14-mer peptides derived from the sequence of HIV reversed transcriptase. A second mode of operation allows the synthesis to be carried out on the surface of polyethylene pins. Peptides derived from the sequence of human TNF were synthesized using this method and used to characterize antibodies raised against the intact protein.
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PMID:Automated multiple peptide synthesis. 128 42

Philadelphia-chromosome positive chronic myeloid leukemia cells in chronic phase (CML-CP) or blast crisis (CML-BC) and normal bone marrow cells (NBMC) were incubated in vitro with antisense oligonucleotide specific against the BCR/ABL breakpoint junction to examine the possibility of selective inhibition of leukemia growth. Growth capability was determined in vitro by colony assay in semisolid medium in the presence of interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF). The 18-mer antisense directed against the specific BCR/ABL mRNA breakpoint region diminished the colony formation by CML-CP and CML-BC cells, but not by NBMC. Scrambled oligomer did not affect significantly the growth of leukemic and normal cells. If CML-BC cells were mixed with NMBC and incubated with specific BCR/ABL antisense oligomer, leukemic colonies were selectively inhibited, as was shown by reverse, transcriptase-polymerase chain reaction (RT-PCR) performed to detect BCR/ABL mRNA in single colonies. These results confirm the possibility of selective inhibition of leukemia cells by antisense treatment.
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PMID:Gene-targeted specific inhibition of chronic myeloid leukemia cell growth by BCR-ABL antisense oligodeoxynucleotides. 179 39

The catalytic properties of electrophoretically homogeneous RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue were studied with the aid of oligonucleotides of defined sequence. It was found that RdRP catalyzes in vitro the transcription of short single-stranded RNA and DNA molecules into precisely complementary RNA copies up to the full length of these templates. The transcription of RNA- and DNA-oligonucleotide templates was equally effective. Differences in transcription efficiency were found to depend on nucleotide sequence rather than on the RNA or DNA nature of the single-stranded nucleic acid. Double-stranded nucleic acids such as poly(A).poly(U) and a double-stranded DNA 14-mer were not transcribed. The RdRP-directed transcription could be primed because RNA and DNA dinucleotides and trinucleotides complementary to the 3'-terminal nucleotides of the template were extended by the enzyme. The unprimed transcription was shown to start preferentially at the 3'-terminal nucleotides of the template. RdRP is capable of adding a single noncomplementary nucleotide to the 3' terminus of about 50% of the runoff transcripts. AMP was preferred over GMP, whereas CMP and UMP were terminally added at very low frequency.
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PMID:RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties. 768 23

RNA replication, a process of fundamental importance for pathogenesis by many viruses, remains poorly understood at the mechanistic level because relatively few of the responsible enzymes have been purified and characterized biochemically. Partially purified RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley leaves is able to synthesize (-)-strand RNAs from input (+)-strand templates. In resolving RdRp products generated during (-)-strand BMV RNA synthesis, we found that an RNA of eight nucleotides was generated at approximately 10-fold molar excess to the full-length (-)-strand RNA. Production of the 8-mer was dependent upon and specific to BMV RNA templates. Furthermore, inhibitors of full-length (-)-strand RNA synthesis by RdRp also affected the production of the 8-mer. Analysis of the sequence of the 8-mer suggests that it is initiated at the authentic initiation site on the BMV RNA template, consistent with the possibility that the 8-mer is an abortive initiation product of RNA synthesis by RdRp in vitro. Addition of heparin or Mn2+ differentially affected production of the 8-mer and full-length (-)-strand RNA, as did some nucleotide changes near the site of RNA initiation. These studies further define the initiation process of (-)-strand RNA synthesis.
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PMID:Initiation of (-)-strand RNA synthesis catalyzed by the BMV RNA-dependent RNA polymerase: synthesis of oligonucleotides. 894 17

The association of increased metallothionein (MT) gene expression in breast cancer with metastasis and poor prognosis has led us to investigate the hypothesis that inhibition of MT gene expression may elicit antiproliferative effects in breast carcinoma MCF7 cells. To monitor the effect of downregulation of MT protein on growth, MCF7 cells were transiently transfected by electroporation with an 18-mer MT antisense phosphorothioate oligomer (AO) or an 18-mer random oligomer (RO). The MT-AO is complementary to the region 7 bases downstream from the AUG translational start site of the hMT-IIA gene. Transfection of MCE7 cells with the AO inhibited cell growth by 50-60% at 72 hours when compared to control cells or the cells transfected with RO. The AO-induced growth inhibition was associated with alterations in morphology suggestive of apoptotic cell death. This was further confirmed by DNA linker cleavage into oligonucleosomal fragments and decreased bcl-2 protein levels in AO-transfected cells as opposed to the RO-transfected cells. Reverse transcriptase polymerase chain reaction analysis showed that AO induced a 2-fold increase in the levels of c-fos and p53 transcripts in comparison to RO which had no significant effect. Conversely, c-myc transcripts were decreased by 2.5-fold in the AO-transfected cells when compared to the controls. Furthermore, MCF7 cells transfected with an expression plasmid pBAcNEO-sMT-IIA encompassing human MT-IIA cDNA, constitutively driven by beta-actin promotor, caused a 2.5-fold increase in intracellular levels of MT, as judged by PCR and western blot analysis, in comparison to the cells transfected with pBAcNEO plasmid. In contrast to the AO-induced growth inhibition, overexpression of cytoplasmic MT increased the cell multiplication by 2-fold compared with control cells or the cells transfected with the control plasmid 72 hours post-transfection. Moreover, the effects of AO on oncogene expression were reversed on increased expression of MT. These data suggest that overexpression of MT potentiates the growth of MCF7 cells, whereas downregulation of MT elicits antiproliferative effects.
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PMID:Antisense down-regulation of metallothionein induces growth arrest and apoptosis in human breast carcinoma cells. 917 39

Quantitative competitive reverse-transcriptase PCR is the most sensitive method for studying gene expression. To investigate whether the accuracy of the calculated target mRNA copy number is affected by the cDNA priming process, we utilized primers of different lengths, concentrations and primer sequences to prime cDNA synthesis reactions. Our results show a approximately 19-fold increase in the calculated mRNA copy number from cDNA synthesis reactions primed with random hexamers (P<0.001, n=4), and a approximately 4-fold increase in copy number with a specific hexamer (P<0.001, n=4) compared with that obtained with a 22-mer-sequence-specific primer. The increase in calculated mRNA copy number obtained by priming cDNA synthesis with the shorter specific and non-specific primers could be explained largely by the synthesis of truncated standard cDNA molecules lacking a requisite binding site for amplification with PCR primers. Since these truncated standard cDNA molecules could not be amplified and standard RNA is used to quantify target mRNA copy number, this phenomenon resulted in overestimation of target mRNA copy number. In conclusion, accurate determination of target mRNA copy number is most likely if a long specific antisense primer is used to prime cDNA synthesis.
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PMID:Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR. 988 20

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

The RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene has been expressed as a nonfusion protein in bacterial cells and purified to homogeneity using sequential chromatographic columns. The purified NS5B protein exhibited RNA-dependent RNA polymerase activity using poly(A) template and the K(m) and V(max) were determined as 8.4 microM and 1976 pmol/mg-min, respectively. This full-length NS5B protein exhibited much stronger binding affinity toward the 30-mer poly(G) than other homopolymeric RNAs of the same size. For the first time, we demonstrate that the HCV NS5B was able to bind various ribonucleotides. Using a panel of oligonucleotides varying in length, we studied the NS5B catalytic efficiency and proposed the size of the NS5B active site to be 8-10 nucleotides. The multifunctional nature of NS5B protein is also discussed and compared with other viral RNA polymerases.
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PMID:Specificity and mechanism analysis of hepatitis C virus RNA-dependent RNA polymerase. 1077 51

The non-structural protein 5b (NS5b) of hepatitis C virus (HCV), bearing an RNA-dependent RNA polymerase (RdRp) activity, is considered as a new target of antiviral therapy. We expressed and purified the C-terminal 21 amino acid truncated NS5b protein fused with glutathione S-transferase (GST-5bC21) using Escherichia coli. With the highly purified GST-5bC21 protein, we established an in vitro assay system for RdRp activity by using poly(C) as the template and a 12 mer oligo(rG) as the primer. The optimal conditions for testing various concentrations of template, primer and proteins were determined to 22 degrees C and a pH of 7.5. The addition of 2.5 mM Mn(2+) increased the activity profoundly, to a level fivefold higher than that in the presence of 10 mM Mg(2+). At higher concentrations of Mn(2+), GST-5bC21 is stable as compared with previously reported full-length NS5b expressed using insect cells or NS5b protein with the C-terminal 18 amino acids deleted. This sensitive and easy to use quantitative assay system will provide a stable system for the screening of inhibitors for HCV RdRp.
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PMID:Measurement of HCV RdRp activity with C-terminal 21 aa truncated NS5b protein: optimization of assay conditions. 1204 62

De novo RNA synthesis by hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3'-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that 3'-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the +1, +2, or +3 position numbered from the 3' end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the +2 and +3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the +1 position and to maintain the integrity of the 3' end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.
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PMID:Selection of 3'-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependent RNA polymerase. 1207 3


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