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)

Past work has shown that transformed Escherichia coli is not a suitable vehicle for studying the expression and regulation of the cloned luminescence (lux) genes of Vibrio harveyi. Therefore, we have used a conjugative system to transfer lux genes cloned into E. coli back into V. harveyi, where they can be studied in the parental organism. To do this, lux DNA was inserted into a broad-spectrum vector, pKT230, cloned in E. coli, and then mobilized into V. harveyi by mating aided by the conjugative plasmid pRK2013, also contained in E. coli. Transfer of the wild-type luxD gene into the V. harveyi M17 mutant by this means resulted in complementation of the luxD mutation and full restoration of luminescence in the mutant; expression of transferase activity was induced if DNA upstream of luxC preceded the luxD gene on the plasmid, indicating the presence of a strong inducible promoter. To extend the usefulness of the transfer system, the gene for chloramphenicol acetyltransferase was inserted into the pKT230 vector as a reporter. The promoter upstream of luxC was verified to be cell density regulated and, in addition, glucose repressible. It is suggested that this promoter may be the primary autoregulated promoter of the V. harveyi luminescence system. Strong termination signals on both DNA strands were recognized and are located downstream from luxE at a point complementary to the longest mRNA from the lux operon. Structural lux genes transferred back into V. harveyi under control of the luxC promoter are expressed at very high levels in V. harveyi as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis: the gene transfer system is thus useful for expression of proteins as well as for studying the regulation of lux genes in their native environment.
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PMID:Transcriptional regulation of lux genes transferred into Vibrio harveyi. 218 Sep 15

We studied a clinical isolate of Salmonella typhi (strain 1895) characterized by resistance to 200 micrograms of chloramphenicol per ml despite the absence of chloramphenicol-inactivating activity. The outer membrane protein profile analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a deficiency of one of the major protein species which may serve as a porin for entry of chloramphenicol. When the strain was transformed with a plasmid encoding chloramphenicol acetyltransferase, chloramphenicol added to the culture was not inactivated, suggesting a drastic reduction of permeability towards the drug. Moreover, transformants bearing a plasmid coding for the Escherichia coli OmpF porin became considerably more susceptible to chloramphenicol (40 micrograms/ml). On the other hand, transformants carrying a plasmid encoding the Salmonella typhi ompC gene remained as resistant to the drug as the parental strain, even though they overexpressed OmpC. These findings indicate that the lack of OmpF plays a major role in the resistance to chloramphenicol in strain 1895.
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PMID:Clinical isolate of a porinless Salmonella typhi resistant to high levels of chloramphenicol. 228 83

Anaerobic bacteria currently demonstrate increased resistance to antimicrobial agents, primarily by the production of beta-lactamase. A number of species of Bacteroides, most notably those in the Bacteroides fragilis group, produce these enzymes. A few species of Fusobacterium and Clostridium produce beta-lactamase as well. Fortunately, this mechanism of resistance is readily overcome by administering beta-lactamase inhibitors coupled with a beta-lactam antibiotic that would otherwise be inactivated. Other types of resistance encountered in anaerobic bacteria include inactivating enzymes such as chloramphenicol acetyltransferase, plasmid-mediated transferable multiple-drug resistance, changes in porin molecules in the outer membrane of the bacterial cell, decreased uptake of drug by other mechanisms, changes in the target organs such as penicillin-binding proteins, and decreased reduction of the antibiotic to an active intermediate product. In many institutions, certain drugs such as cefoxitin, clindamycin, and piperacillin, which were previously active against almost all strains of B. fragilis, are now effective against only 70 to 85% of this group of anaerobes. Drugs with essentially 100% activity against most anaerobic bacteria include chloramphenicol, imipenem, metronidazole, and the combinations of a beta-lactam antibiotic plus a beta-lactamase inhibitor such as ampicillin plus sulbactam and amoxicillin or ticarcillin combined with sodium clavulanate. This paper also discusses the indications for antimicrobial susceptibility testing of anaerobes as well as problems encountered with testing techniques that are currently being used.
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PMID:Mechanisms of resistance in anaerobes and new developments in testing. 268 14

To study the effect of sodium butyrate on human immunodeficiency virus (HIV) long terminal repeat (LTR)--directed expression, we constructed a chimeric plasmid (pLTR-CAT) in which the LTR sequences derived from a molecular clone of HIV were fused to the chloramphenicol acetyltransferase (CAT) gene. We used transient expression assays in transfected tissue culture cells to monitor the activity of the LTR. The expression of the pLTR-CAT plasmid was activated when the cells were exposed to butyrate after transfection. The magnitude of butyrate-induced increase was linear up to an 8 mM concentration and was different with regard to the target promoters used. Recombinant plasmids linked to marker genes may be useful models for studying the effects on HIV of various agents of chemical and biological origin.
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PMID:Sodium butyrate activates human immunodeficiency virus long terminal repeat--directed expression. 282 93

A recombinant vaccinia virus that expresses the human immunodeficiency virus (HIV) trans-activator (tat) gene was constructed. The tat polypeptide migrated anomalously with an apparent molecular mass of 14 kDa on a sodium dodecyl sulfate-polyacrylamide gel and reacted with polyclonal anti-tat serum. The tat protein was localized predominantly in the cell nucleus despite the absence of other HIV proteins or intranuclear HIV DNA. Additional recombinant vaccinia viruses that contain the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under control of an early vaccinia promoter were constructed. Insertion of the HIV trans-activator-responsive (tar) sequence at the precise start of the CAT mRNA decreased CAT expression slightly. Trans-activation of vaccinia virus-encoded tarCAT failed to occur when CV-1 or HeLa cells were coinfected with the recombinant vaccinia virus expressing tat or when a HeLa cell line containing stably integrated copies of tat was used for infection, indicating the absence of transcriptional or translational effects under these conditions.
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PMID:Use of vaccinia virus vectors to study the synthesis, intracellular localization, and action of the human immunodeficiency virus trans-activator protein. 283 62

In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.
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PMID:Induction of gene expression under human cytomegalovirus immediate early enhancer-promoter control by inhibition of protein synthesis is cell cycle-dependent. 283 70

HPV-6vc subgenomic fragments were inserted into an enhancer-dependent expression vector for chloramphenicol acetyltransferase (CAT) and assayed for the presence of transcriptional enhancing elements. A transcriptional enhancing element was detected in the noncoding region (NCR) of the HPV-6vc viral genome when the CAT assays were performed in viral transformed human kidney cell lines (293 and 324K), in human cervical carcinoma cell lines (HeLa and Siha), and in bovine papillomavirus type 1 (BPV-1) transformed mouse cells (C127-53). The NCR region of the HPV-6b genome was only capable of enhancing transcription of the CAT gene in the HeLa cell line at a level one-third that of the HPV-6vc NCR. The HPV-6vc NCR enhancing activity in C127-53 cells was further stimulated by the addition of sodium butyrate to the growth medium. Localization of the DNA sequences in the HPV-6vc NCR responsible for enhancing transcription revealed two distinct enhancer elements. One element (HPV-6vc position 7218-7544) was active in the 293, HeLa, Siha, and C127-53 cells. The second enhancer element (HPV-6vc position 7544-7971) was only capable of stimulating transcription in HeLa, C127-53, and Siha cells. When the HPV NCR-CAT expression vectors were cotransfected with a competitor plasmid (pNCR75) into C127-53 or HeLa cells then transcriptional enhancement decreased, indicating competition of cellular factors which affect both segments of the HPV-6vc NCR.
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PMID:The noncoding region of HPV-6vc contains two distinct transcriptional enhancing elements. 302 99

The cotransfection of selectable marker genes and the gene for the nonstructural proteins NS1 and NS2 of the autonomous parvovirus H-1 failed to produce cell lines that constitutively expressed NS1. A plasmid, pP38NS1cat, was constructed that expressed the NS1-NS2 gene from the H-1 P38 coat protein promoter in place of the natural P4 promoter. The P38 promoter is constitutively weak and is trans-activated by NS1. Stable cell lines were isolated that contained pP38NS1cat that was constitutively silent, but inducible with exogenous NS1 by superinfection or by treatment with sodium butyrate. The cells that were induced for this self-stimulatory genetic circuit did not remain in the culture, suggesting that expression of NS1-NS2 is cytotoxic or that the expression is not sustained. The properties of these cell lines and an example of the construction of a cell line inducible for expression of the viral coat protein gene and the bacterial gene for chloramphenicol acetyltransferase (cat) are described.
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PMID:Construction of a genetic switch for inducible trans-activation of gene expression in eucaryotic cells. 303 74

Rat cell lines were established in which the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) was stably integrated. The cell lines showed a repressed phenotype for CAT expression, but could be induced for it by inhibition of protein synthesis, as well as by heat-shock and chemical inducers of the cellular stress response, such as sodium arsenite, 8-hydroxyquinoline and the heavy metals cadmium and copper. A decameric sequence present in the NF-kB binding sites in the HIV LTR (GGGACTTTCC) resembles the cellular heat-shock core sequence and may therefore be involved in the heat-shock response.
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PMID:Heat-shock induction of the human immunodeficiency virus long terminal repeat. 318 32

We have developed a modified, reproducible, and efficient method for introducing cloned genes into mammalian cells by using an electric field followed by treatment with sodium butyrate. Transfection frequencies with plasmid pSV2-neo, consisting of an antibiotic (G418) resistance gene and simian virus 40 (SV40) early promoter, by electroporation were higher than those by calcium phosphate DNA precipitation. Treatment with sodium butyrate following electroporation significantly increased the frequency of transfection in various types of cell lines and primary cultured cells including human skin fibroblasts. Treatment with sodium butyrate also increased the transient expression of the gene for chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol O3-acetyltransferase, CAT, EC 2.3.1.28) when the gene was introduced into BALB/c 3T3 cells by electroporation. Electroporation combined with sodium butyrate treatment is an improved method for stable and transient biochemical transformation of foreign genes in cultured mammalian cells.
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PMID:An improved method of electroporation for introducing biologically active foreign genes into cultured mammalian cells. 340 76


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