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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.
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PMID:Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus. 154 75

Anti-Fc gamma R IgM monoclonal antibodies (mAbs) isolated from lipopolysaccharide-stimulated spleen cells from tightskin (TSK) mice were found to be polyspecific, reacting with a wide variety of molecules, including double-stranded DNA, topoisomerase, RNA polymerase, and different collagen types. Approximately 60% of the polyspecific IgM mAbs have anti-Fc gamma R specificity. These anti-Fc gamma R mAbs induce the release of hydrolases from both azurophil and specific granules of human neutrophils. 25-45% of the total cellular content (determined in Nonidet P-40 lysates) of neutrophil elastase, 10-25% of beta-glucuronidase, and 30-50% of alkaline phosphatase was released after incubation with the mAbs. The degranulation process was accompanied by dramatic morphological changes shown by scanning and transmission electron microscopy. The release of hydrolytic enzymes stimulated by the IgM anti-Fc gamma R mAbs was inhibited by preincubation of neutrophils with Fab fragments of either anti-human Fc gamma RII (IV.3) or anti-human Fc gamma RIII (3G8) mAbs. The binding of the anti-Fc gamma R TSK mAbs to human neutrophils was inhibited by Fab fragments of mAb 3G8. However, we found that the TSK anti-Fc gamma R mAbs do not bind to human Fc gamma RII expressed in either CHO cells or the P388D1 mouse macrophage cell line. Since the enzyme release could be inhibited by Fab fragments of mAb IV.3, we suggest that the signal transduction may require Fc gamma RII activation subsequent to crosslinking of the glycan phosphatidyl inositol-anchored Fc gamma RIII-1. These data demonstrate for the first time that polyspecific autoantibodies with Fc gamma R specificity can trigger neutrophil enzyme release via human Fc gamma RIII-1 in vitro and indicate a possible role for such autoantibodies in autoimmune inflammatory processes.
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PMID:IgM anti-Fc gamma R autoantibodies trigger neutrophil degranulation. 182 27

We have identified and partially characterized a complex transcriptional unit within the murine beta-glucuronidase gene complex on chromosome 5. On the same strand and within the first intron of the beta-glucuronidase structural gene, Gus-s, we observe an RNA polymerase II promoter motif. That sequences within this carefully defined region can promote RNA polymerase II transcription is supported by results of in vitro transcriptional runoff assays and by expression of a linked reporter gene in both cultured cells and transgenic mice. Results of RNA blot hybridization and S1 nuclease protection studies reveal a 2.2-kilobase processed liver transcript which is initiated just downstream of the promoter motif and sharing little, if any, sequence with the 2.7-kilobase beta-glucuronidase mRNA. Both RNA species are found in liver where beta-glucuronidase is known to be expressed in all cell types. To our knowledge, this is the first description of eukaryotic mRNAs from overlapping transcription units which share the same strand yet exhibit little, if any, sequence similarity. A possible regulatory relationship between these overlapping structural genes is discussed.
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PMID:Overlapping transcriptional units on the same strand within the murine beta-glucuronidase gene complex. 318 71

Alanine and phenylalanine tRNA sequences were amplified by PCR from Arabidopsis thaliana nuclear DNA using degenerate oligonucleotides which introduced specific mutations into the acceptor stem. The aminoacylation of T7 RNA polymerase transcripts of these sequences was investigated in vitro using partially purified bean alanyl- or phenylalanyl-tRNA synthetase. In parallel, the in vivo activity of amber suppressor derivatives of these tRNAs was investigated in transient expression assays in tobacco protoplasts using a beta-glucuronidase (GUS) reporter gene containing a premature amber stop codon. The results show that mutation of the G3:U70 base pair to G3:C70 blocks aminoacylation of plant alanine tRNA, whilst conversion of the G3:C70 pair normally found in plant tRNA(Phe) to G3:U70 enables the mutated tRNA(Phe) to be a good substrate for alanyl-tRNA synthetase and impairs its aminoacylation with phenylalanine. In addition, the amber suppressor derivative of wild-type tRNA(Phe) showed very little suppressor activity in vivo, and was poorly aminoacylated with phenylalanine in vitro, suggesting that the anticodon is a major identity determinant for tRNA(Phe) in plant cells.
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PMID:Characterization of some major identity elements in plant alanine and phenylalanine transfer RNAs. 753 29

The functional architecture of the proximal region of a rice phenylalanine ammonia-lyase (PAL) promoter was analyzed by transcription of PAL-beta-glucuronidase (GUS) templates by whole-cell extracts of rice cell suspension cultures. The promoter 5' truncated to position -35 was sufficient for accurate initiation of basal transcription. Substitution of the TATTTAA sequence between positions -35 and -28 with GCGGGTT or 2-bp substitutions to give TCGTTAA and TATGGAA inactivated the minimal promoter. Moreover, the function of the TATTTAA sequence was dependent on its position relative to the initiation site; hence, this element is an authentic TATA box essential for RNA polymerase II-dependent transcription. Substitutions in the TCCAAG initiator cis element (-3 to +3) at the -1 (C to A or G) and +1 (A to C or T) residues caused inaccurate initiation, whereas mutations at the other residues of this conserved element or sequence substitutions between the TATA box and initiator had little effect. TATA box and initiator functions were confirmed by analysis of the effects of promoter mutations on expression in stably transformed rice cell suspensions and plants. We concluded that the proximal region of the PAL promoter has a simple functional architecture involving a TATA box appropriately positioned upstream of the initiator. Transcription of derivatives of such minimal promoters by highly active cell extracts should allow molecular analysis of functional interactions between specific cis elements and cognate trans factors.
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PMID:TATA box and initiator functions in the accurate transcription of a plant minimal promoter in vitro. 758 Feb 58

A convenient in vitro transcription system using monocot and dicot whole cell extracts and circular DNA templates has been developed. The system consists of incubating template and whole cell extract to generate initiation complexes, followed by addition of nucleotide triphosphates to support elongation, and primer extension assay to detect authentic transcripts. This in vitro transcription system required circularized templates and was essentially inactive with linearized templates. Accurate in vitro transcription of a rice phenylalanine ammonia-lyase (PAL) promoter-beta-glucuronidase (GUS) gene fusion and a tobacco sesquiterpene cyclase promoter-GUS gene fusion was examined in their homologous whole cell extracts, and the optimal concentrations for several reaction components, including DNA template, whole cell extract, monovalent and divalent cations, were determined for specific initiation from the in vivo start site. Transcription was inhibited by low concentrations of alpha-amanitin, demonstrating that the reaction was mediated by RNA polymerase II. Accurate transcription initiation was dependent on the TATA-box motif within the respective promoters. Based on the effect of delayed addition of sarkosyl at a concentration sufficient to inhibit transcription initiation but not elongation, three to four rounds of transcription were initiated in standard assays.
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PMID:Accurate in vitro transcription from circularized plasmid templates by plant whole cell extracts. 759 45

An auxin-regulated gene, parA, comprises a gene family consisting of a handful genes which respond to various signals. Although Droog et al. (Plant Mol. Biol, 1993, 21, 965-972) postulated that the parA-related genes belong to the family of a cytoplasmic enzyme, glutathione S-transferase (GST), we detected a low level of GST activity in the parA products, whose value was below 1/30 of that of parB products encoding tobacco (Nicotiana tabacum L.) GST. Immunofluorescence studies using an antibody against parA protein revealed that the subcellular location of parA protein is the nucleus in cultured tobacco mesophyll protoplasts, while conventional GSTs' including the parB product were primarily located in the cytoplasm. Confocal laser scanning microscopy of tobacco BY-2 cells showed that the parA product was confined to the nucleus, but was excluded from the nucleolus. In addition, exon/intron organization of the parA family was appreciably different from that of conventional GSTs including parB. Furthermore, the parA protein is much more similar to a 24-kDa protein of Escherichia coli that is reported to bind to RNA polymerase. These different characteristics of parA compared with to the conventional GSTs, indicate that parA protein would have distinct functions, such as involvement in transcription, rather than functioning as a conventional GST. Transgenic tobacco plants that carried the parA promoter fused to a beta-glucuronidase gene were used to show that the parA gene is tissue-specific and also under developmental control.
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PMID:Expression of the auxin-regulated parA gene in transgenic tobacco and nuclear localization of its gene products. 776 32

Phage T7 RNA polymerase has been used extensively in Escherichia coli for high-level expression of selected genes placed under the control of the phage T7 gene 10 promoter. We have constructed an analogous system for use in plastids of higher plants. A T7 RNA polymerase chimeric gene containing a cauliflower mosaic virus 35S promoter and a tobacco ribulose-bisphosphate carboxylase/oxygenase small-subunit chloroplast transit-peptide sequence was introduced into tobacco by nuclear transformation. Stable plastid transformation of tobacco expressing the T7 RNA polymerase activity with a T7 promoter/beta-glucuronidase (GUS) reporter gene construct resulted in expression of GUS mRNA and enzyme activity in all tissues examined. Expression of GUS activity was extremely high in mature leaves, moderate in young leaves and petals, and low in stems, roots, and developing seeds. Plastid transformation of wild-type tobacco with the same chimeric GUS gene resulted in undetectable levels of GUS mRNA and enzyme activity. Genetic crosses demonstrated that a silent T7/GUS reporter gene could be activated in the F1 generation by transmission of an active nuclear T7 RNA polymerase gene from the male parent.
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PMID:Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. 804 84

In Flaveria pringlei, a C3 plant, P protein of the glycine-cleavage system is encoded by a small gene family consisting of at least five transcriptionally active genes. We have cloned and sequenced two of these genes, gdcsPA and gdcsPB, and provide the first detailed report on the complete structure of eukaryotic gdcsP genes. Based on the lengths of exons and intervening sequences, the P-protein genes can be subdivided into two parts. In both cases the N-terminal region consists of one very long exon followed by a long intron. In contrast, the C-terminal parts show a complex mosaic structure of relatively small exons and introns. A highly conserved leucine-zipper motif was identified, which is supposed to participate in the assembly of the glycine decarboxylase multienzyme complex. The transcript derived from the gdcsPA sequence corresponds perfectly to a leaf cDNA isolated earlier. Reverse-transcriptase PCR experiments show that both genes are preferentially active in leaves. Stems contain distinctly less P protein mRNA and the relative level in roots is very low but still clearly detectable. In all three organs, but most significantly in roots, the gdcsPA transcript level is distinctly higher than that of gdcsPB. Analysis of promoter-beta-glucuronidase fusions in transgenic tobacco suggests that far-upstream elements enhance the transcriptional activity of both genes in leaves relative to stems. The analysis of distal gdcsPA promoter deletions reveals the presence of regulatory elements acting with a distinct organ preference and indicates their approximate location.
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PMID:Structure and expression analysis of the gdcsPA and gdcsPB genes encoding two P-isoproteins of the glycine-cleavage system from Flaveria pringlei. 852 30

We have developed a new T-DNA vector, pGA2715, which can be used for promoter trapping and activation tagging of rice (Oryza sativa) genes. The binary vector contains the promoterless beta-glucuronidase (GUS) reporter gene next to the right border. In addition, the multimerized transcriptional enhancers from the cauliflower mosaic virus 35S promoter are located next to the left border. A total of 13,450 T-DNA insertional lines have been generated using pGA2715. Histochemical GUS assays have revealed that the GUS-staining frequency from those lines is about twice as high as that from lines transformed with the binary vector pGA2707, which lacks the enhancer element. This result suggests that the enhancer sequence present in the T-DNA improves the GUS-tagging efficiency. Reverse transcriptase-PCR analysis of a subset of randomly selected pGA2715 lines shows that expression of the genes immediately adjacent to the inserted enhancer is increased significantly. Therefore, the large population of T-DNA-tagged lines transformed with pGA2715 could be used to screen for promoter activity using the gus reporter, as well as for creating gain-of-function mutants.
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PMID:T-DNA insertional mutagenesis for activation tagging in rice. 1248 Oct 47


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