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)

A full-length cDNA clone of barley yellow dwarf virus (BYDV-PAV serotype) has been constructed and fused to the bacteriophage T7 RNA polymerase promoter. RNA transcripts produced in vitro, either capped or uncapped, were infectious in Triticum monococcum protoplasts. Protoplasts inoculated with in vitro-transcribed BYDV RNA accumulated coat protein, synthesized new viral RNAs, and produced virus particles. Aphid feeding on extracts from protoplasts inoculated with in vitro RNA transcripts can be used to transfer the virus progeny to whole plants. Introduction of mutations which interrupt specific BYDV-PAV open reading frames (ORFs) V and VI eliminated infectivity while an ORF I-mutant remained infectious. Infectious RNA transcripts derived from BYDV cDNA clones will facilitate analysis of the molecular aspects of BYDV infection and further enhance our understanding of this economically important virus.
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PMID:Infectious in vitro transcripts from a cloned cDNA of barley yellow dwarf virus. 198 57

The 35S rRNA gene of the yeast Saccharomyces cerevisiae was fused to the GAL7 promoter. This hybrid gene, when present on a multicopy plasmid and induced by galactose, suppressed the growth defects of a temperature-sensitive RNA polymerase I (pol I) mutant and those of a mutant in which the gene for the second largest subunit of pol I was deleted. Analysis of pulse-labeled RNA directly demonstrated that rRNA synthesis in this deletion mutant is from the GAL7 promoter. These experiments show that the sole essential function of pol I is the transcription of the rRNA genes, that pol I is not absolutely required for the synthesis of rRNA and ribosomes or cell growth if 35S rRNA synthesis is achieved by some other means, and that the tandemly repeated structure of the chromosomal rRNA genes is also not absolutely required for the synthesis of rRNA and ribosomes.
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PMID:Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I. 202 44

The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two essentially nonhomologous ssRNAs of 3.9 kb (RNA-1) and 1.45 kb (RNA-2) which are each capped at the 5' terminus with m7GpppA. cDNA clones short of full length by several nucleotides at both termini have been generated to both RNAs. Oligonucleotide-directed mutagenesis was employed to generate a series of RNA-1 and -2 transcription vectors in which the bacteriophage T7 RNA polymerase promoter was fused to full-length cDNA clones. Yields of in vitro transcripts initiating with wild-type viral 5'-terminal adenosine were extremely low. Efficient transcription was achieved only when one, or alternatively two, nonviral guanosines were engineered 5' to the authentic viral sequence at the transcription start site. m7GpppG-capped or -uncapped RCNMV RNA-1 and RNA-2 transcripts were infectious and induced symptoms identical to those of wild-type virus infection when coinoculated on the systemic hosts Nicotiana benthamiana and N. clevelandii, and on the local lesion host Chenopodium amaranticolor. Uncapped in vitro transcripts were somewhat less infectious. Progeny virus derived from infectious transcript inoculum was as infectious as wild-type virus. Primer extension analysis indicated that the 5'-terminal nonviral guanosine residues were not maintained in the progeny virus.
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PMID:Red clover necrotic mosaic virus infectious transcripts synthesized in vitro. 202 74

Human brain hexokinase (hexokinase I) was produced in Escherichia coli from a synthetic gene under control of the bacteriophage T7 promoter. The expressed coding region derives from a human cDNA clone thought to specify hexokinase I based on amino acid sequence identity between the predicted translation product and hexokinase I from rat brain. The open reading frame from this cDNA was fused to the promoter and 5' flanking region of T7 gene 10, and expressed in E. coli by induction of T7 RNA polymerase. Induced cells contained a hexokinase activity and an abundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to near homogeneity consisted of a 100,000 Da protein, the size predicted from the nucleotide sequence of the expressed cDNA. The purified enzyme had Michaelis constants of 32 microM and 0.3 mM for glucose and ATP, respectively, and bound to rat liver mitochondria in the presence of MgCl2. Enzymatic activity was inhibited by glucose-6-P and this inhibition was relieved by inorganic phosphate. Deinhibition by phosphate is a property specific to brain hexokinase.
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PMID:Expression of human brain hexokinase in Escherichia coli: purification and characterization of the expressed enzyme. 204 17

Site-directed mutagenesis was used to examine the organization of cis-acting regulatory elements that comprise the promoter of the early 35,000-molecular-weight protein gene (35K protein gene) encoded by the EcoRI-S region of the baculovirus Autographa californica nuclear polyhedrosis virus. The promoter fragment, extending from positions -226 to +12 relative to the early RNA start site (position +1), was fused to the reporter gene encoding chloramphenicol acetyltransferase (CAT) and then inserted into the genome of recombinant viruses (3.96 map units) in order to ascertain the role of regulatory elements in the context of a normal infection. A combination of deletions and linker insertions revealed that early transcription was mediated by a basal (minimum) promoter, consisting of the TATA element (positions -30 to -25), that was in turn responsive to an upstream activating region located between -90 and -30. The TATA element exerted the single greatest influence on the level of early promoter activity and contained all information necessary to direct transcription from a site located 30 nucleotides downstream. The upstream activating region provided a 10- to 15-fold stimulation of transcription from the early +1 start site that was mediated by distinct DNA elements. These regulatory elements included two GC motifs (centered at positions -81 and -54, respectively), composed of alternating G and C residues, and a CGT motif (position -40) that contained the core sequence A(A/T)CGT(G/T). Each motif was required for full promoter activity during the early phase of infection. This organization that employs diverse cis-acting stimulatory elements is typical of promoters responsive to RNA polymerase II and may facilitate increased expression of A. californica nuclear polyhedrosis virus genes early in infection when the level of viral DNA for transcription is critically low.
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PMID:Identification of upstream promoter elements mediating early transcription from the 35,000-molecular-weight protein gene of Autographa californica nuclear polyhedrosis virus. 207 43

Steroid receptors have been reported to stimulate transcription in a manner synergistic with other transcription factors. We have examined this synergism or functional cooperativity between glucocorticoid receptors and basal transcription factors in a variety of promoter and reporter gene contexts. A fragment containing a hormone response element from mouse mammary tumor virus was fused to well characterized promoters from the herpes virus thymidine kinase and mouse beta-globin genes and to related mutant promoters altered by inactivation of transcription factor-binding sites through point mutagenesis or deletion. These constructs were transfected into glucocorticoid-sensitive fibroblasts, and reporter gene activity was assessed with or without hormonal stimulation. In contrast to previous studies, we found little indication of synergistic interaction between elements mediating a hormone response and adjacent basal promoters. In fact, we observed that inactivating basal factor-binding sites, thereby decreasing promoter strength, actually increased hormone inducibility. We suggest that the inverse relationship between basal promoter strength and the induction ratio attained upon hormonal stimulation may be due to limitation of a common factor, an "adaptor" through which glucocorticoid receptor and basal transcription factors interact with the components of the RNA polymerase II complex to stimulate rates of transcription.
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PMID:Concerted stimulation of transcription by glucocorticoid receptors and basal transcription factors: limited transcriptional synergism suggests mediation by coactivators/adaptors. 207 21

We have fused the ribosomal RNA promoter P1 from the rrnB gene of Escherichia coli to lacZ and examined its guanosine tetraphosphate (ppGpp)-dependent expression at different growth rates. The rrnB P1-lacZ fusion was constructed on plasmid vectors and then recombined into the chromosome of a delta lac relA1 strain close to the normal location of the rrnB locus and in the normal orientation, coincident with the direction of replication. A series of spoT strains differing in the severity of their SpoT defect were analyzed with respect to their growth characteristics and ppGpp metabolism. The spoT203 allele was introduced into the rrnB P1-lacZ fusion containing strain and used to manipulate the ppGpp concentration independently of the growth medium. 1) The levels of ppGpp during exponential growth are decreased in rich media due to a decreased activity of (p)ppGpp synthetase II (PSII). 2) The specific activity of rrn P1-directed beta-galactosidase was increased in a parabolic fashion with increasing growth rate. A theoretical analysis showed that this was to be expected since enzyme expression from a stringently controlled promoter is affected by changes in the growth rate both via the control of the promoter, and indirectly via the control of bulk mRNA synthesis. 3) The observer specific enzyme activities were converted into rrnB P1 promoter activities, given as lacZ transcription relative to the total rate of transcription. The rrn P1 promoter activity decreased exponentially with increasing cytoplasmic concentration of ppGpp, independent of whether a given level of ppGpp was achieved by using different growth media or by using a spoT allele. These results support two hypotheses: (i) that RNA polymerase is partitioned by ppGpp into two fractions with different abilities to initiate transcription at rrn P1 promoters; and (ii) that during exponential growth ppGpp is derived from ppGpp synthetase II (PSII). Together these hypotheses predict that the growth rate control of rRNA synthesis reflects a control of PSII activity which is regulated by the composition of the growth medium.
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PMID:Guanosine tetraphosphate (ppGpp) dependence of the growth rate control of rrnB P1 promoter activity in Escherichia coli. 211

We have expressed two cDNA sequences encoding 121 and 230 amino acids of the C-terminus of the Schistosoma mansoni Hsp 70 in Escherichia coli. The products were synthesized as polypeptides fused to the RNA polymerase of bacteriophage MS2, and their reactivities were tested in ELISAs, using sera from human and murine infections. Anti-Hsp70 antibodies were detected in a significant number of individuals suffering from chronic schistosomiasis mansoni, but not in patients with known recent infections. This, together with the finding that antibodies directed at S. mansoni-specific Hsp70 determinants during the course of infection of experimental mice were not detectable until 5-6 weeks post-infection, suggests that the protein may be a useful marker for distinguishing late and early infections. The diagnostic specificity of Hsp70 was evaluated with sera from humans infected with different schistosome species and other parasitic diseases. While some subjects infected with S. haematobium produced antibodies which recognized the S. mansoni Hsp70, no such antibodies were generated in S. japonicum infected individuals. However, cross-reactive antibodies were elicited in donors with other parasitic diseases such as filariasis and malaria. The absence of antibodies in early infection and the observed cross-reactivities led us to conclude that Hsp70 will be of limited value in the diagnosis of schistosomiasis.
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PMID:The humoral response to heat shock protein 70 in human and murine Schistosomiasis mansoni. 211 91

We found that the 5' nontranslated leader sequence from encephalomyocarditis virus (EMCV) allowed transcripts that were synthesized by the T3 RNA polymerase in mammalian cells to be translated in a cap-independent fashion. Stable mouse cell lines that carry the T3 RNA polymerase gene expressed the chloramphenicol acetyltransferase (CAT) gene under the control of a phage promoter when the CAT gene was fused to the EMCV leader and introduced into the cells by transient DNA uptake. The level of gene expression in such cells was similar to or greater than that observed with a conventional transient expression vector that is dependent on transcription by the host RNA polymerase II. Expression of the EMCV-CAT fusion gene was stimulated by cotransfection of the cells with a gene that encodes the poliovirus protease 2A protein (which inhibits cap-dependent translation), demonstrating that the EMCV-CAT fusion gene was expressed in a cap-independent fashion. Introduction of both the T3 RNA polymerase gene and the EMCV-CAT fusion gene into a variety of cultured mammalian cell lines (HeLa, BSC40, Ltk-, NIH 3T3, and C127) demonstrated that the T3-EMCV expression system functions in a broad range of cell types.
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PMID:Synthesis of functional mRNA in mammalian cells by bacteriophage T3 RNA polymerase. 216 33

The human ADH1, ADH2, and ADH3 genes are closely related members of a gene family which are differentially expressed during liver development. To begin examining the mechanism of this tissue-specific and stage-specific expression, the 5'-flanking nucleotide (nt) sequences of the three genes were determined and the transcription start point (tsp) were identified. Sequences of all three genes indicated a high degree of homology (greater than 80% nt sequence identity) from the AUG translation start codon to about nt -780 relative to the tsp. Transient transfection assays of a set of plasmids containing various lengths of ADH 5'-flanking DNA fused to cat were performed in the HepG2 and Hep3B human hepatoma cell lines. The results indicated that the ADH2 promoter-proximal region was transcriptionally active in the absence of upstream sequences. To identify potential cis-acting elements in the ADH2 promoter-proximal region, a DNase I footprinting assay using a rat liver nuclear extract was used. Protection occurred in several locations including one, between nt -51 and -10, which shares homology with known binding sites for a previously identified rat-liver transcription factor called CCAAT/enhancer binding protein (C/EBP). Purified C/EBP was shown by footprint analysis to bind at two distinct sites in the ADH2 promoter located at nt -51 to -31 and -21 to -10. The TATA-box promoter element at nt -30 to -22 was not protected by C/EBP, but was partially protected by a factor in the rat liver nuclear extract. Thus, it is possible that the flanking C/EBP molecules may create a novel binding pocket for TFIID, the TATA-binding general transcription factor for RNA polymerase II. Alternatively, the C/EBP molecules may block access to the TATA box, and stimulate transcription of ADH2 by interacting with some component(s) other than TFIID.
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PMID:Promoters for the human alcohol dehydrogenase genes ADH1, ADH2, and ADH3: interaction of CCAAT/enhancer-binding protein with elements flanking the ADH2 TATA box. 216 44


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