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)

To examine the RNA polymerase (EC 2.7.7.6) specificity of RNA maturation/utilization and transcriptional enhancement, we constructed a chimeric plasmid (pPolI-CAT) in which a promoter for mouse rRNA gene transcription was placed adjacent the coding sequences for chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). A number of other constructs, including plasmids also containing a murine sarcoma virus enhancer or lacking any natural eukaryotic promoter sequences, were also prepared. In apparent agreement with earlier conclusions that an RNA polymerase I transcript can act as a messenger RNA, transient transfection of mouse L cells with pPolI-CAT yielded both high levels of transcription from the RNA polymerase I promoter and enzymatically active CAT protein. However, further examination revealed that CAT protein is not translated from RNA that begins at the normal rRNA transcription initiation site. Polysomal RNA is devoid of such RNA and instead consists of CAT-encoding transcripts that begin elsewhere in the mouse ribosomal DNA (rDNA) region. Since transcription of these aberrant RNAs is stimulated by the addition of a murine sarcoma virus enhancer segment, they are probably transcribed by RNA polymerase II. Transcripts that map to the authentic rRNA start site are not similarly enhanced. Moreover, unlike the RNAs deriving from the rRNA initiation site, these aberrant RNAs are more stable and the level of translatable CAT transcripts is suppressed by inclusion of larger segments of the rDNA promoter regions. Fortuitously initiated mRNAs are also formed in the absence of any natural eukaryotic promoter sequence. From these data we conclude that there is no evidence that normal RNA polymerase I transcription yields functional mRNA and that transcriptional enhancement appears to be RNA polymerase specific.
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PMID:RNA polymerase specificity of mRNA production and enhancer action. 346 18

The nucleotide sequence of the inducible chloramphenicol acetyltransferase gene (cat) of Staphylococcus aureus plasmid pC221 has been determined. The deduced primary structure for the 215 residue polypeptide (25.9 kDa) is in agreement with partial amino acid sequence data on the purified protein, previously designated as the type C variant of CAT. In common with the inducible cat elements of pC194 and B. pumilus, the 5' non-coding region of the cat of pC221 contains an inverted complementary repeat ('stem-loop' or 'hairpin') which may sequester the predicted ribosome bonding site of the mRNA. The likely transcription initiation site has been determined in vitro using purified B. subtilis RNA polymerase. Recombinant plasmids carrying the cat of pC221 on a 1156 bp TaqI fragment are expressed inefficiently in Escherichia coli, wherein induction is both poor and orientation-specific.
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PMID:Chloramphenicol acetyltransferase gene of staphylococcal plasmid pC221. Nucleotide sequence analysis and expression studies. 385 95

Nucleotide sequences of 188 promoter-containing DNA regions have been studied by the computer statistic analysis. Undecanucleotide NTT(G/C)TTGACA(A/T) or (G/C) X TT(G/C)A(G/C)A(A/T)TT(G/T) (recognition site) and heptanucleotide RTATATR or TATAATR (initiation site) separated by 12-19 base pairs are characteristic of a "generalized" promoter structure. Promoters can function if a minimal level of correspondence for their recognition and initiation sites to a generalized structure is attained (the correspondence function value for the whole structure is not lower than 0,61; for the most effective promoters it may be equal to 1). The transcription start is situated 3-9 base pairs after initiation site, 4-7 pairs distance being the most effective. Transcription can start from any nucleotide, preferably with A or G. The start from A is the most effective if it is contained within the CAC or CAT trinucleotides. The promoter efficiency is enhanced by some additional structural factors: the presence of an extended A-T rich region directly before the recognition site; availability of integral promoter structures or several RNA polymerase binding sites in the preceding nucleotide sequence. A characteristic feature of the promoter is the presence of either the dyadic axial symmetry elements in the initiation and recognition sites as well as in the intermediate region, or the A-T rich area in the latter.
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PMID:[Statistical study of the nucleotide sequence of a prokaryotic promoter. Structural elements determining the efficacy of the transcription initiation stages]. 623 57

Peptides of melanosomal proteins have recently been shown to be recognized in an HLA-restricted mode by specific cytolytic T lymphocytes in melanoma patients. Dendritic antigen-presenting cells (DC) are considered to be the most effective stimulators of T cell responses, and the use of these cells has therefore been proposed to generate therapeutic responses to tumor antigens in cancer patients. We, therefore, generated DC from peripheral blood of normal donors in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4. Flow cytometric analysis of the cells during a 2-week culture revealed a loss of CD14 and CD34 expression, a concomittent increase of CD1a, CD11a,b and c, CD44, CD45, CD54, HLA-class I and II, and intermediate levels of CD26, CD80 and CD86. Cultured DC stimulated proliferation of allogeneic T cells and induced a marked, up to 20-fold, stimulation of T cell proliferation after pulsing with tetanus toxoid. To achieve independence of already-identified antigenic peptides presented in HLA class I-restricted fashion, which limits the general applicability of such peptides for vaccination of melanoma patients, we tested whether DC are transfectable with eukaryotic expression plasmids. DC transfected with two reporter genes (CAT, beta-galactosidase) using a liposome-based transfection technique, exhibited only low levels of enzymatically active proteins, but were able to degrade rapidly intracellular proteins and to process peptides efficiently. Chloramphenicol acetyltransferase as well as tyrosinase mRNA were detectable after transfection by reverse-transcriptase-polymerase chain reaction, and enzyme activities became measurable. Furthermore, DC transfected with the tyrosinase gene were able to induce specific T cell activation in vitro, indicating appropriate peptide processing and presentation in DC after transfection. These data suggest new approaches to future tumor vaccination strategies.
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PMID:Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation. 748 49

An established cell line, clone 64, in which the expression of the RNA polymerase PB1 and PA subunit genes and the nucleoprotein (NP) gene but not the PB2 subunit gene of influenza virus can be induced by the addition of dexamethasone, was used to analyze the replication and transcription machineries of the influenza virus. Both NS-CATc and NS-CATv, the chimeric nonstructural protein chloramphenicol acetyltransferase (NS-CAT) RNAs in the sense and antisense orientations positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8 (the NS gene), respectively, can be transcribed into the corresponding complementary-strand RNA in clone 64 cells only when treated with dexamethasone. Although sense-strand poly(A)+ CAT RNA was detected in the dexamethasone-treated clone 64 cells transfected with NS-CATv RNA, CAT activity was not detected in these cells and the isolated poly(A)+ CAT RNA was inert in an in vitro translation system. However, when the poly(A)+ CAT RNA was capped by using a purified yeast mRNA capping enzyme (mRNA guanylyltransferase), the capped poly(A)+ CAT RNA became translatable in the in vitro translation system. These results indicated that PB1, PA, and NP can support the replication of the influenza virus genome as well as the transcription to yield uncapped poly(A)+ RNA and that PB2 is specifically required for the synthesis of capped RNA.
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PMID:The RNA polymerase PB2 subunit is not required for replication of the influenza virus genome but is involved in capped mRNA synthesis. 781 36

A new temperature-regulated T7 RNA polymerase-driven transcription system has been developed. This system is based on a hybrid regulatory region: the phage T7 late promoter (PT7) linked to the Escherichia coli lac operator (Olac) [Giordano et al., Gene 84 (1989) 209-219], which was located in an earlier obtained [Mashko et al., Gene 97 (1991) 259-266] temperature-controlled amplifiable plasmid, carrying cat under the control of PT7-Olac and, in addition, lambda major early promoter-operator regions and gene cIts857. Plasmids of the pT7-Olac-cat-tsr series were stably maintained at a low-copy-number when grown at low temperature (28 degrees C). In E. coli BL21(DE3), carrying the Plac-controllable T7 RNA polymerase-encoding gene, efficient repression of cat transcription was observed, that was provided by the LacI repressor and, probably, the thermolabile repressor CIts857. At low and moderate temperatures (28/37 degrees C), this 'cooperative' repression was so tight that cat expression was not observed in the cells carrying PT7-Olac on the plasmids, even after IPTG-inducible T7 RNA polymerase biosynthesis. As a result of the thermo-amplification of the recombinant plasmids and temperature-inactivation of CIts857, expression of the T7 RNA polymerase-encoding gene was derepressed due to the titration of LacI by the increasing copies of Olac which in turn, led to the highly efficient T7 RNA polymerase-driven accumulation of CAT in the cells.
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PMID:A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator. 818 58

This paper describes the cytotoxicity of ranunculin (RAN) and its mechanism of action. The IC50 of RAN against the KB and Bel7402 cells in colony test were found to be 0.21 and 0.35 mumol/L respectively. RAN inhibited the incorporation of 3H-labeled precursors into DNA and RNA of L1210 cells. RAN (15 mumol/L) markedly decreased DNA synthesis catalyzed by DNA polymerase I and promoted the generation of superoxide anions in DMSO/KO2 system. In the meantime, SOD and CAT were shown to partly revoke the inhibitory effects of RAN upon the incorporation of 3H-TdR into DNA. No direct reaction between RAN and DNA template was observed and no effect of RAN on DNA TOPO II or RNA polymerase was found. Our results suggest that the cytotoxicity of RAN in vitro may be due to inhibition of DNA polymerase and increase of oxygen free radicals.
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PMID:[The cytotoxicity and action mechanism of ranunculin in vitro]. 823 75

Expression of bacteriophage T7 RNA polymerase in mammalian cells can efficiently drive the transcription of a foreign gene controlled by the T7 promoter (Elroy-Stein et al., Proc. Natl. Acad. Sci. USA. 86, 6126-6130, 1989). We have tested the hypothesis that purified T7 RNA polymerase can be co-delivered into mammalian cells together with a reporter gene (chloramphenicol acetyltransferase, CAT) controlled by the T7 promoter (pT7-EMC-CAT) using DC-chol cationic liposomes. Indeed, significant level of CAT activity was observed in human lung adenocarcinoma (A549-1) cells which had been incubated with a complex of T7 RNA polymerase, pT7-EMC-CAT DNA and DC-chol cationic liposomes. The expression was specific in that T3 RNA polymerase could not replace the T7 RNA polymerase, and that co-delivered T7 RNA polymerase did not enhance the expression of a CAT gene controlled by the SV40 early promoter. The system was optimized in terms of enzyme, DNA and liposome concentrations. Time course experiment indicated that the expression of the T7 system was about 8-10 hours sooner than the SV40 system, consistent with the notion that T7 RNA polymerase does not enter into the nucleus and the transcription takes place in the cytoplasm of the transfected cells. The expression of the T7 system was transient; it declined after 30 hours post transfection, probably due to turnover of the phage enzyme in the mammalian cells. The expression system described here should be useful for gene transfer experiments which require a fast but transient expression of a foreign gene. We have also compared our delivery system with a commercial reagent, Lipofectin, which has been used to deliver T3 or T7 RNA polymerase with a reporter plasmid encoding the T3 or T7 promoter.
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PMID:Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes. 833 95

The compatible plasmids pKGP1-1 and pCM-X# will confer chloramphenicol resistance to Escherichia coli harboring the two plasmids if the T7 RNA polymerase produced from pKGP1-1 can recognize the T7 promoter carried on pCM-X# and transcribe the CAT gene that is cloned behind the promoter [Ikeda et al. (1992) Biochemistry 31, 9073-9080]. When E. coli harbor pKGP1-1 and a pCM-X# plasmid that carries a point mutation in the T7 promoter that destroys promoter activity (an inactive pCM-X#), the T7 RNA polymerase will not utilize the T7 promoter point mutant, will not produce CAT, and will not induce chloramphenicol resistance. The selection of mutants of T7 RNA polymerase that exhibit altered promoter recognition was pursued by randomly mutagenizing pKGP1-1 with aqueous hydroxylamine, cotransforming E. coli with the mutagenized pKGP1-1 and a mixture of seven different inactive pCM-X# plasmids, and isolating and characterizing the RNA polymerase that was present in those colonies that exhibited chloramphenicol resistance. It was established that E. coli harboring the mutant plasmid pKGP-HA1mut4 and an inactive pCM-X# are chloramphenicol-resistant and that the mutation responsible for the expression of CAT from the inactive pCM-X# plasmid is a G to A transition at nucleotide 664 of T7 gene 1 that converts glutamic acid (222) to lysine. Apparently this mutation expands the range of T7 promoter sequences that can be utilized by the enzyme. The mutant T7 RNA polymerase, GP1(Lys222), utilizes all seven inactive T7 promoter point mutants more efficiently than wild-type T7 RNA polymerase both in vivo and in vitro. Furthermore, the correlation of in vivo and in vitro promoter utilization suggests that the restoration of chloramphenicol resistance in the cotransformed E. coli results from the ability of GP1(Lys222) to initiate transcription from T7 promoter point mutants that are normally inactive.
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PMID:Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences. 836 83

hnRNP protein A1 (34 kDa, pl 9.5) is a prominent member of the family of proteins (hnRNP proteins) that associate with the nascent transcripts of RNA polymerase II and that accompany the hnRNA through the maturation process and the export to the cytoplasm. New evidence suggests an active and specific role for some of these proteins, including protein A1, in splicing and transport. Contrary to the other hnRNP proteins, the intracellular level of protein A1 was reported to change as a function of proliferation state and cell type. In this work we analyse the A1 gene expression in different cells under different growth and differentiation conditions. Proliferation dependent expression was observed in lymphocytes and fibroblasts while purified neurons express high A1 mRNA levels both in the proliferative (before birth) and in the quiescent (after birth) state. Transformed cell lines exhibit very high (proliferation independent) A1 mRNA levels compared to differentiated tissues. A structural and functional characterization of the A1 gene promoter was carried out by means of DNase I footprinting and CAT assays. The observed promoter features can account for both elevated and regulated mRNA transcription. At least 12 control elements are contained in the 734 nucleotides upstream of the transcription start site. Assays with the deleted and/or mutated promoter indicate a co-operation of multiple transcriptional elements, distributed over the entire promoter, in determining the overall activity and the response to proliferative stimuli (serum).
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PMID:Human hnRNP protein A1 gene expression. Structural and functional characterization of the promoter. 838 72


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