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)

The molecular mechanism of interferon action on vaccinia virus-specific immediate early protein synthesis was studied in interferon-treated chick cells. In line with previous observations, the synthesis of total vaccinia WR virus-specific mRNA, thymidine kinase (TK) mRNA, and several other early mRNAs was detectable by short [3H]uridine pulses. Under conditions of over 90% inhibition of poxvirus-specific TK induction, accumulation of TK mRNA was strongly inhibited. Northern blot analysis revealed strong degradation of residual TK mRNA prepared from interferon-treated chick embryo fibroblasts (CEF). Blot hybridization analysis using total vaccinia DNA and restriction fragment N as probes demonstrated a generally reduced steady-state amount of vaccinia virus-specific early mRNAs in interferon-treated CEF. When CEF were infected with a recombinant vaccinia virus strain into the TK gene of which the chloramphenicol acetyltransferase gene had been inserted, CAT activity was far lower in interferon-treated than in untreated CEF. We conclude that signals that specify rapid breakdown of viral TK mRNA in interferon-treated CEF are located in the regions flanking the coding sequences of the viral TK gene.
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PMID:Reduced steady-state levels of vaccinia virus-specific early mRNAs in interferon-treated chick embryo fibroblasts. 243 97

We are investigating the feasibility of using the positive-strand RNA virus Sindbis virus and its defective interfering (DI) particles as vectors for introducing foreign genes into cells. In previous work we showed by deletion mapping of a cloned cDNA derived from one of the DI RNAs that only nucleotides at the 3' and 5' termini of the RNA are essential for the DI RNA to be amplified after it is transfected into cells in the presence of helper virus. As a first step in developing a vector we replaced 75% of the internal nucleotides of this DI cDNA with foreign sequences including the bacterial chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. DI RNAs transcribed from this cDNA were replicated and packaged by helper Sindbis virus and became a major viral RNA species in infected cells by the third passage after transfection. They were also translated to produce enzymatically active CAT. CAT activity was measured at passage 3 but could also be detected in transfected cells. DI RNAs containing the CAT gene were translated in vivo and in vitro to produce two polypeptides immunoprecipitable by anti-CAT antibodies. One polypeptide was identical in size to the authentic CAT polypeptide; the other was the size expected for a protein initiated at an upstream, viral-specific AUG in frame with the CAT AUG. These studies establish that DI genomes of Sindbis virus can tolerate the insertion and direct the expression of at least one foreign gene.
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PMID:Engineered defective interfering RNAs of Sindbis virus express bacterial chloramphenicol acetyltransferase in avian cells. 244 30

To determine the block(s) to spleen necrosis virus (SNV) replication in mouse cells, we studied the expression of a dominant selectable marker, neo, or a gene whose product is easily assayed, the chloramphenicol acetyltransferase (cat) gene, in SNV-derived and murine leukemia virus-derived vectors. Using transient (CAT) and stable (Neor phenotype) transfection assays, we showed that the SNV promoter was used in mouse cells only when the 3' SNV long terminal repeat (LTR) was absent. Infection of mouse cells with recombinant SNV viruses was 1% as efficient as infection of permissive dog (D17) cells. The SNV proviruses in mouse cells appeared normal by Southern blot analysis, indicating that their integration probably occurred by normal mechanisms. S1 nuclease analyses of Neor mouse cell clones, each harboring a single recombinant SNV provirus, showed that the selected (internal) promoter was active, but that the 5' SNV LTR promoter was not. However, in the rare (less than 10(-6)) Neor colonies in which expression of the 5' LTR was selected, both promoters were active. Thus, the block to SNV infection of mouse cells is at least at two levels; one is a 100-fold-decreased efficiency at some step(s) up to and including integration, and the other is at transcription.
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PMID:Transcription from a spleen necrosis virus 5' long terminal repeat is suppressed in mouse cells. 244 16

Primary murine keratinocytes can be maintained in culture for extended periods in a proliferative, basal cell state under conditions of reduced extracellular Ca2+. In response to increased Ca2+ concentrations, the cells undergo a well-defined program of terminal differentiation, thus serving as a convenient model in which to study the genes involved in regulating this and possibly other differentiation cascades by DNA-mediated gene transfer. However, because of their sensitivity to increased Ca2+ concentrations, the introduction of exogenous genomic DNA into primary keratinocytes by conventional methods is problematic. We have optimized the calcium phosphate DNA transfection procedure by introducing conditions that reduce the potency of Ca2+ as a differentiation signal. Primary epidermal cells were transfected with pSV2CAT, a plasmid that codes for the enzyme chloramphenicol acetyltransferase CAT. Enzyme activity was measured in cell extracts under varying transfection conditions. When the K+ concentration of the medium used for transfection by calcium phosphate precipitation is reduced from 6.5 to 0.01 mM, CAT activity following transfection increases 2-3 times. Exposure to the DNA precipitate for 2-4 h is optimal. By the use of fibroblast conditioned medium following transfection, enzyme activity can be detected in cell extracts for at least 21 d, suggesting that the exogenous gene is integrated. The low K+/Ca2+ transfection method is more effective than SrCl2 used as an alternative for CaCl2 in Ca2+ sensitive cells. Low K+ medium enhances cell survival for Ca2+ mediated transfection but also appears to have a beneficial effect on DNA uptake or expression.
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PMID:Expression of transfected DNA by primary murine keratinocytes. 245 57

The level of alpha-fetoprotein (AFP) mRNA in HuH-7 human hepatoma cells is elevated by the addition of dexamethasone to the culture medium. To locate the DNA region involved in hormonal regulation of the AFP gene, we constructed recombinant plasmids in which various lengths of the 5'-flanking sequence of the human AFP gene were fused to the CAT gene. Various cell lines were transfected with the recombinant plasmids, incubated with or without 3 x 10(-6) M dexamethasone, and then assayed for chloramphenicol acetyltransferase expression. In hepatoma cells that produce AFP, the dexamethasone treatment resulted in the stimulated chloramphenicol acetyltransferase expression when the transfected plasmids contained 169 base pairs (bp) or longer AFP 5'-flanking sequence. No dexamethasone effect was observed when the 5'-flanking sequence was less than 98 bp long. The dexamethasone stimulation was effectively suppressed by the glucocorticoid antagonist RU486, indicating that this effect is mediated by glucocorticoid receptors. The 71-bp region between positions -169 and -98 contains a nucleotide stretch which is similar to the consensus sequence of the glucocorticoid responsive element (GRE). Partial alterations of this sequence resulted in decreased dexamethasone response. The GRE-containing region stimulated heterologous (SV40) promoter activity in response to dexamethasone treatment in an orientation- and position-independent manner. The GRE and the upstream AFP enhancer function independently from each other.
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PMID:Transcriptional regulation of alpha-fetoprotein expression by dexamethasone in human hepatoma cells. 246 58

We located and characterized transcription terminators in the E1a and E1b genes by transferring the 3' fragments of the genes into the vector pSCAT10 [Sato et al., Mol. Cell. Biol. 6 (1986) 1032-1043] at a site located immediately downstream from the cat gene (coding for chloramphenicol acetyltransferase; CAT) and upstream from the simian virus 40 polyadenylation region. Multiple terminators were located downstream from the E1b gene, but not in the 3' region of the E1a gene. Fine analysis of these terminators by the CAT assay method and S1 nuclease mapping of in vivo transcripts indicated possible involvement of a G-rich sequence in transcription termination of the E1 region. These terminators were repeated tandemly and used by both the E1a and the E1b genes in a orientation-dependent manner.
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PMID:Multiple transcription terminators in E1a and E1b genes of adenovirus type 5. 246 6

The 5'-region of the rat alpha 2-macroglobulin gene has been characterized. A 5.6 kb Sal I - Xba I fragment containing the first 4 exons of the alpha 2-macroglobulin gene and 1.3 kb of its 5'-flanking region was sequenced. The putative transcriptional start site was determined by RNase protection and primer extension analysis. TATA- and CAAT-box equivalent sequences were found. A potential glucocorticoid receptor binding site was located on the antisense strand. DNA sequences containing the 5'-flanking region of the rat alpha 2-macroglobulin gene were linked to the gene coding for the bacterial chloramphenicol acetyltransferase and introduced into Hep G2 cells. In these transfected Hep G2 cells CAT activity could be induced by recombinant human interleukin-6. Deletion analyses have shown that the sequences between -852 and -777 as well as between -404 and -165 relative to the cap site, contain regulatory elements involved in the interleukin-6 dependent induction of the alpha 2-macroglobulin gene.
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PMID:Identification of the promoter sequences involved in the interleukin-6 dependent expression of the rat alpha 2-macroglobulin gene. 246 33

pAFP-CAT, a recombinant plasmid containing 5'-flanking sequence from -7 kb to +7 bp of rat alpha-fetoprotein (AFP) gene can drive the expression of the bacterial chloramphenicol acetyltransferase gene in McA-RH7777 and McA-RH8994 rat hepatoma cell lines. Dexamethasone treatment suppresses pAFP-CAT expression in McA-RH7777 cells but increases its expression in McA-RH8994 cells, which mimics the dexamethasone responses of the endogenous AFP gene in both cell lines. However, dexamethasone treatment enhanced pMMTV-CAT expression in both cell lines. These data suggest that the effects of dexamethasone on AFP gene expression may be mediated by different trans-acting factors binding to the specific cis-elements of the 5'-flanking region of the rat AFP gene.
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PMID:The mechanism of the bidirectional regulation of the rat alpha-fetoprotein gene by glucocorticoid hormone. 247 82

The zif268 gene, which encodes a protein with three typical zinc finger sequences, is induced in mouse 3T3 cells by serum, phorbol 12-myristate 13-acetate platelet-derived growth factor, and fibroblast growth factor. The induction is coordinate with that of c-fos. The 5'-flanking region of zif268 contains sequences that resemble known regulatory elements, including four CC(A or T)6GG sequences similar to the core serum response elements (SREs) found upstream of c-fos and actin genes. To determine whether the zif268 SRE-like elements mediate induction, CAT (chloramphenicol acetyltransferase) plasmids with different lengths of zif268 upstream sequences were tested for inducibility in 3T3 cells by serum, platelet-derived growth factor, or phorbol 12-myristate 13-acetate. In addition, double-stranded oligonucleotides corresponding to each of the four zif268 putative SREs were tested individually for responsiveness when placed upstream of a thymidine kinase gene promoter. Each of the four SREs conferred inducibility by the agents tested, and multiple SREs resulted in greater inducibility than did a single element. Each of the zif268 SREs also competed with the c-fos SRE for binding by serum response factor present in HeLa cell nuclear extract. We conclude that the zif268 SRE-like sequences are functional and probably account for the coordinate induction of zif268 and c-fos.
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PMID:Functional serum response elements upstream of the growth factor-inducible gene zif268. 251 79

A 1.2-kilobase pair fragment of the 5' upstream region of a potato wound-inducible gene (wun1) was fused to different marker genes (wun1-CAT, wun1-NPTII). Stable integration of a wun1-CAT chimeric gene into the tobacco genome led to a high wound-inducible chloramphenicol acetyltransferase activity in leaves. Transient expression experiments in potato protoplasts showed that wun1 carries a strong promoter sequence similar in strength to the 35S promoter. The same intensity of expression was also observed using wun1 constructs in transient experiments with rice protoplasts. wun1 mRNA was shown to accumulate to high levels in potato leaves collapsing as a result of infection with the phytopathogen Phytophthora infestans. The wun1 product might, therefore, play a role in a general physiological reaction to stress correlated with cell death.
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PMID:5' upstream sequences from the wun1 gene are responsible for gene activation by wounding in transgenic plants. 253 62


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