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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glucocorticoids inhibit transcription of the murine cytoplasmic thymidine kinase gene (Tk-1). Glucocorticoid regulation of Tk-1 transcription can be demonstrated in cells that are arrested in late G1. This observation indicates that inhibition of Tk-1 expression is not dependent upon redistribution within the cell cycle but is due to glucocorticoid regulation of this gene. Transfection studies have been carried out using chimeric genes in which restriction fragments of the Tk-1 promoter were fused to chloramphenicol acetyltransferase or neomycin phosphotransferase. These chimeric reporters were assayed for stable expression and glucocorticoid regulation in P1798 lymphoma cells. A 140-bp fragment, extending from -143 to -3 bp with respect to the thymidine kinase translational start site, was capable of both basal and glucocorticoid-regulated transcription of reporter genes. The extent of inhibition by glucocorticoids was similar to that observed for the endogenous gene, and no increase in basal expression or the extent of inhibition was observed with constructs containing additional 5'-flanking DNA. The 140-bp Tk-1 core promoter fragment binds to transcription factors in extracts from P1798 cells. Control cell extracts contain factors that bind to and protect (from deoxyribonuclease I) a distal promoter element from -106 to -87 bp, relative to the translational start site. A second, proximal element was protected at -43 to -36 bp. The proximal element of the Tk-1 promoter resembles an RNA polymerase II initiator element. No other elements were protected. Glucocorticoids inhibit the amount or activity of the transcription factor that binds to this initiator-like element within the Tk-1 promoter. This element, when fused to upstream activation sequences from the herpes simplex virus thymidine kinase promoter, conveys glucocorticoid sensitivity in cis.
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PMID:Glucocorticoid regulation of a transcription factor that binds an initiator-like element in the murine thymidine kinase (Tk-1) promoter. 896 Dec 64

The La autoantigen is an RNA-binding protein that is involved in initiation and termination of RNA polymerase III transcription. It also binds several viral RNAs, including those of poliovirus and human immunodeficiency virus (HIV). Binding of the La protein to these RNAs enhances their translation in vitro (K. Meerovitch, Y.V. Svitkin, H.S. Lee, F. Lejbkowicz, D.J. Kenan, E.K.L. Chan, V.L. Agol, J.D. Keene, and N. Sonenberg, J. Virol. 67:3798-3807, 1993, and Y.V. Svitkin, A. Pause, and N. Sonenberg, J. Virol. 68:7001-7007, 1994). Here, a functional domain in the carboxy-terminal half of La that is distinct from the RNA-binding domain is described. Deletion of this domain abrogated the ability of La protein to enhance translation of poliovirus RNA and a hybrid HIV trans-activation-response element-chloramphenicol acetyltransferase mRNA. Far-Western assays indicated that the La protein homodimerized in vitro, and the C-terminal deletions that caused a loss of activity in translation also abrogated the dimerization signal. Gel filtration chromatography of recombinant La protein confirmed that La protein exists as a dimer under native conditions. Addition of the purified dimerization domain resulted in a loss of translation stimulatory activity of La protein in cell-free-translation reactions.
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PMID:The La autoantigen contains a dimerization domain that is essential for enhancing translation. 897 96

RNA polymerase I transcription in vivo in transiently DNA-transfected cells has been used to express influenza virus vRNA molecules coding for chloramphenicol acetyltransferase (CAT) in an antisense orientation. Influenza virus superinfection provided viral RNA polymerase and other proteins required for transcriptional conversion of minus-strand vRNA into plus-strand viral mRNA molecules expressing CAT activity. This system has been used for analysis of the vRNA sequences which cooperatively constitute the vRNA promoter structure via nucleotide exchanges as well as deletions and insertions of both terminal segments. Several mutants caused greatly enhanced expression over wild-type levels, which was transmitted during serial passage of progeny virus. The data obtained for the mutations in various promoter elements support a model implicating double-stranded vRNA promoter structures in binding of viral polymerase, and in consecutive steps during initiation of RNA synthesis.
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PMID:Mutational analysis of influenza virus promoter elements in vivo. 904 76

Nitrate increases the transcription of the two Arabidopsis thaliana nitrate reductase genes. We demonstrated previously that 238 and 330 bp of the 5' flanking regions, designated as NP1 and NP2, of the two nitrate reductase genes NR1 and NR2, respectively, are sufficient for nitrate-dependent transcription (Y. Lin, C.-F. Hwang, J.B. Brown, C.-L. Cheng [1994] Plant Physiol 106: 477-484). Here we identify the cis-acting elements of NP1 and NP2 that are necessary for nitrate-dependent transcription by linker-scanning (LS) analysis. In transgenic plants one LS mutant of NP1 and two LS mutants of NP2 exhibited significantly lower nitrate-induced reporter gene chloramphenicol acetyltransferase activity. To distinguish which of these three mutants lost nitrate inducibility, competitive reverse-transcriptase polymerase chain reaction was used to measure the chloramphenicol acetyltransferase mRNA levels before and after nitrate induction. The single LS mutant in NP1 lost its response to nitrate, whereas the two LS mutants in NP2 partially lost their response to nitrate. A 12-bp sequence is conserved between the NP1 site and the two NP2 sites. This sequence motif is also conserved in the 5' flanking regions of other nitrate-inducible plant genes. Gel mobility shift experiments indicate that these three regions bind to similar proteins. The binding is constitutive with respect to nitrate treatment and was observed in both nonphotosynthetic suspension cells and green leaves.
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PMID:Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes. 908 75

We have developed plasmid-based expression systems that encode modified forms of T7 RNA polymerase (RNAP) having 6-12 histidine residues fused to the amino terminus. The histidine-tagged RNAPs (His-T7 RNAPS) are indistinguishable from the wild-type (WT) enzyme in nearly all biochemical assays. Similar plasmids that encode His-tagged T3 and SP6 RNAPs have also been constructed. To facilitate site-directed mutagenesis of the RNAP gene, the size of the target plasmid was minimized by using T7 RNAP itself as a selectable marker. BL21 (DCAT4) cells (which carry a chromosomal copy of the chloramphenicol acetyltransferase cat gene under control of a T7 promoter) are resistant to chloramphenicol when functional T7 RNAP is expressed, thus allowing the selection and maintenance of the target plasmid in these cells. Mutagenesis is accomplished by denaturing the plasmid, annealing mutagenic DNA primers, and repairing the plasmid with T4 DNA polymerase. Two DNA primers are used: one corrects a defect in the bla gene, the other introduces the desired mutation into the RNAP gene; 30-85% of the ampicillin-resistant transformants carry the desired mutation in the RNAP gene. By using BL21 (DCAT4) cells as a recipient for transformation the functional integrity of the RNAP gene may conveniently be monitored by assessing the level of chloramphenicol resistance in vivo. Methods for rapid, simultaneous purification of multiple samples of modified (His-tagged) and conventional RNAPs are described. Together, these developments greatly enhance our ability to characterize this important class of enzymes.
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PMID:Rapid mutagenesis and purification of phage RNA polymerases. 911 96

We have demonstrated that Antisense phosphodiester (ODNs) and phosphorothioate oligonucleotides (S-ODNs) inhibit CAT (chloramphenicol acetyltransferase) protein expression in the clone 76 cell line, which is a derivative of the murine C127 cell line. This cell line expresses the influenza virus RNA polymerase and nucleoprotein (NP) genes in response to treatment with dexamethasone. Phosphodiester, phosphorothioate, and liposomally encapsulated oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The liposomally encapsulated ODNs and S-ODNs complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. On the other hand, the inhibitory effect of the S-ODNs targeted to PB1 was considerably decreased in comparison with the other three target sites. Liposome encapsulation afforded oligomer protection in serum-containing medium and substantially improved cellular accumulation. The liposomally encapsulated oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. Liposomal preparations of oligonucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. The activities of the unmodified oligonucleotides are effectively enhanced by using the liposomal carrier. In the observation of the endocapsulated antisense phosphodiester oligonucleotide, FITC-ODN-PB2-as treated clone 76 cells by a confocal laser scanning microscope, diffuse fluorescence was apparently observed in the cytoplasm. Interestingly, the endocapsulated antisense phosphorothioate oligonucleotide, FITC-S-ODN-PB2-as accumulated in the nuclear region of clone 76 cells. However, weak fluorescence was observed on the endosomes and in the cytoplasmes of the free antisense phosphorothioate oligonucleotides treated clone 76 cells.
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PMID:[Antisense nucleic acid therapy of influenza virus]. 936 Apr 4

Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.
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PMID:Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1. 937 37

We have determined the nucleotide sequences of the regions 3' and 5' proximal to the avian pneumovirus (APV) N and L genes, respectively. These sequences were used in the construction of a synthetic minireplicon construct in which the chloramphenicol acetyltransferase (CAT) reporter gene was flanked at its 3' end with the APV leader together with the APV N gene start signal and at its 5' end with the APV L gene end signal and the genome trailer region. The ability of T7 RNA polymerase runoff transcripts to direct the replication and expression of the CAT reporter gene in APV-infected cells demonstrated the ability of the putative leader and trailer regions to direct genome replication and gene expression. Furthermore, this confirms the absence of the NS1 and NS2 gene analogs within the APV genome. We were able to detect the expression of CAT protein from cells that had been infected with supernatants from the initially infected and transfected cells. These results have identified the cis-acting sequences of APV responsible for viral replication, gene expression, and packaging into virus-like particles.
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PMID:Rescue of synthetic minireplicons establishes the absence of the NS1 and NS2 genes from avian pneumovirus. 937 59

Components of the eukaryotic vaccinia virus/T7 RNA polymerase hybrid expression system were assessed using recombinant and nonrecombinant forms of modified vaccinia Ankara (MVA), a replication-deficient vaccinia virus strain. Recombinant MVA virus expressing T7 RNA polymerase (Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995). Virology 210, 202-205) stimulated high levels of expression from a T7 promoter-chloramphenicol acetyltransferase (CAT) reporter. Most, but not all, of the virally induced expression was T7 RNA polymerase and T7 promoter dependent, with no viral enhancement of translation of T7 transcripts. The efficacy of supplying T7 RNA polymerase expression from nonviral sources was evaluated using a self-amplifying T7 RNA polymerase autogene or an inducible T7 RNA polymerase expression vector. The latter modes yielded CAT activity dependent on T7 RNA polymerase expression; however, expression required viral factors independent of T7 RNA polymerase and did not reach that attained using the recombinant virus. In further experiments, MVA-induced T7 RNA polymerase expression was upregulated by alpha-amanitin, an inhibitor of eukaryotic polymerases. This indicates that MVA/T7 RNA polymerase hybrid expression may be rendered still more efficient by ameliorating transcriptional interference due to an alpha-amanitin-sensitive eukaryotic factor(s).
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PMID:Mechanisms of replication-deficient vaccinia virus/T7 RNA polymerase hybrid expression: effect of T7 RNA polymerase levels and alpha-amanitin. 956 32

Influenza A and B viruses share common sequences and potentially similar panhandle structures in the terminal noncoding regions of virion RNA (vRNA). Interesting differences exist, however, in the number of conserved nucleotides at the 5' and 3' ends of the vRNAs, in base pairs constituting the panhandle duplex, and the length of uridine stretch (U stretch) juxtaposed to the RNA duplex. To analyse the contribution of these signals to the specificity between the two viruses, a transient ribonucleoprotein transfection method was used for the expression of the chloramphenicol acetyltransferase (CAT) reporter gene flanked by the noncoding nucleotides derived from influenza B vRNA. While the base pairing in the RNA duplex was primarily important for template activity, mismatch mutations G11 x G12' and C12 x A13' in the terminal RNA duplex region were utilized by influenza B virus, whereas these mutations were detrimental for influenza A virus. Different activity profiles were observed in the length preference of the RNA duplexes: maximum template activity was observed with 11 base pairs for influenza B virus, and 8 base pairs for influenza A virus. When the mutants with various lengths of U stretch were tested, highest CAT activities were observed with 5 to 7 uridine residues in influenza A virus, whereas in influenza B virus the activity was drastically decreased with 7 uridine residues. We suggest that the specific interaction of influenza virus RNA polymerase with these noncoding cis-acting signals in transcription of the RNA genome, along with unique coding strategies adopted by influenza B virus, has contributed to the divergence of these two closely related viruses.
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PMID:Nucleotides in the panhandle structure of the influenza B virus virion RNA are involved in the specificity between influenza A and B viruses. 956 60


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