Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have constructed a Xenopus laevis oocyte expression vector, pOEV, which allows cloned DNA to be transcribed and translated directly in the oocyte. Since proteins translated in oocytes are post-translationally modified according to conserved eukaryotic signals, these cells offer a convenient system for performing structural and functional analyses of cloned genes. pOEV can be used for direct analysis of proteins encoded by cloned cDNAs without preparing mRNA in vitro, simplifying existing protocols for translating proteins in oocytes with a very high translational yield. Transcription of the vector in oocytes is driven by the promoter for the TFIIIA gene, which can generate 1-2 ng (per oocyte within 2 days) of stable mRNA template for translation. The vector also contains SP6 and T7 promoters for in vitro transcription to make mRNA and hybridization probes. DNA clones encoding chloramphenicol acetyltransferase (CAT) were injected into oocyte germinal vesicles and CAT protein accumulated in the cell over a 2- to 4-day period. We found that the concentration of DNA injected affected protein yields; surprisingly relatively low concentrations in the range 25-50 pg DNA per oocyte gave maximum yields of CAT protein. When as little as 5 pg of pOEV DNA is injected we typically expressed 40 fmol of CAT protein per oocyte, after 4-day incubations. In addition, we have shown that this system is amenable to the expression of nuclear and membrane proteins.
 ...
PMID:pOEV: a Xenopus oocyte protein expression vector. 169 47

A 593 nucleotide fragment of the 5' leader of encephalomyocarditis virus RNA (EMCV-RNA) was linked to the SP6 promoter and inserted upstream of the reporter gene chloramphenicol acetyltransferase (CAT). The presence of the 5'-UTR of EMCV-RNA in the RNA transcripts, made in vitro with the SP6 polymerase, resulted in a strong translational enhancement when tested in the micrococcal nuclease-treated reticulocyte lysate. The transcripts were equally active with or without a 5' methylated capstructure as expected, since EMCV-RNA is one of the mRNAs capable of internal initiation. We searched for a signal in the 5' leader that allows the 43S preinitiation complex to bind internally and localized a hairpin containing a unique nucleotide sequence, CUUUA, present in a domain conserved among cardio- and aphtoviral RNAs. Replacing this sequence into AGCU resulted in a 50% loss of translational activity. A second mutation involving a U-G change in the stem of that hairpin resulted in an almost complete loss of initiation.
 ...
PMID:An initiation signal in the 5' untranslated leader sequence of encephalomyocarditis virus RNA. 216 87

Translation of foreign mRNAs is enhanced by a cis-acting derivative (omega') of the 5'-leader sequence (omega) of tobacco mosaic virus RNA (vulgare strain). To explain this effect we have conducted several experiments in vitro. 1. The presence of various 5'-terminal sequences, including omega', did not significantly increase the half-lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat-germ extract. Also, a long leader sequence, unrelated to omega', did not enhance expression of NPTII mRNA in vitro. 2. The ability of several leader sequences, including omega', to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5'-leader sequence to enhance translation. 3. Expression of CAT mRNA in both wheat germ extract and messenger-dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5'-proximal omega' sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5'-proximal omega' sequence. These data suggest that omega' and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5' terminus more accessible to scanning by 40S ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer-based secondary-structure analyses of our SP6 RNA polymerase transcript sequences. The ability of 5' leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures. A different mechanism probably accounts for the omega'-dependent enhancement of mRNA expression in Escherichia coli or in E. coli cell-free systems.
 ...
PMID:Studies on the mechanism of translational enhancement by the 5'-leader sequence of tobacco mosaic virus RNA. 284 Nov 27

The gene for rat cholecystokinin (CCK) was isolated from a rat genomic DNA library. The transcription unit spans 7 kilobases and is interrupted by two introns. The initiator methionine codon lies 2 bases into exon 2; therefore, exon 1 is a noncoding exon. The transcription initiation site was determined using avian myeloblastosis reverse transcriptase, a cDNA primer, and mRNA isolated from a rat medullary thyroid carcinoma. A "TATA"-like sequence precedes the transcription initiation site at position -34. The polyadenylation site for the gene was mapped by a nuclease protection assay using a cRNA generated by transcription of the exon 3 region of the CCK gene with SP6 bacteriophage RNA polymerase. The sequence AT-TAAA is found 22 bases 5' to the site determined to be the polyadenylation addition site. Two regions of simple repetitive DNA occur within the CCK lambda clone, one within intron 2 and the other 4 kilobases 3' to the gene. Sequence analysis of the repetitive element 3' distal to the gene revealed two copies of the sequence 5'-(AC)n-3', where n is 22 and 25. A 114-base pair sequence of predominantly repeating purine-pyrimidine nucleotides separates these two d(AC) repeats. Transcriptional control elements were investigated by fusing regions of the CCK gene to the structural gene encoding chloramphenicol acetyltransferase. Promoter activity was determined by transfecting COS-7 cells with plasmids containing the gene fusions, followed by determining chloramphenicol acetyltransferase activity in cellular extracts. The region necessary for expression of the CCK gene fusions in COS-7 cells is within 144 bases 5' to the initiation of transcription.
 ...
PMID:A gene encoding rat cholecystokinin. Isolation, nucleotide sequence, and promoter activity. 298 40

Human T-cell leukemia virus type I (HTLV-I) contains the pX sequence which codes for the trans-activator of the long terminal repeat (LTR) and is thus postulated to be associated with leukemogenesis in adult T-cell leukemia. Overlapping open reading frames (ORF) in the pX sequence were recently found to code for p27x-III and p21x-III by ORF III, in addition to p40x coded for by ORF IV. The mechanism of expression of these newly identified proteins and their possible association with trans-activation were studied. On transfection of an expression plasmid that contains a cDNA sequence of the pX mRNA, products from both ORFs III and IV were detected in the cells. The RNA was synthesized in vitro from the cDNA clone by SP6 RNA polymerase and translated in a rabbit reticulocyte lysate. As translation products, two proteins, p27x-III and p21x-III, were detected in addition to p40x. Elimination of the first and second ATG codons in ORF III resulted in loss of the ability to code for p27x-III and p21x-III, respectively, which indicated that the translations from these two ATG codons were independent. A mutant that lacked both ATG codons was fully active in trans-activation of chloramphenicol acetyltransferase gene expression directed by the LTR. These results indicate that a 2.1-kilobase pX mRNA of HTLV-I independently encodes three proteins, p40x, p27x-III, and p21x-III, by different ORFs and that the last two proteins are not involved in trans-activation of the unintegrated LTR.
 ...
PMID:A single species of pX mRNA of human T-cell leukemia virus type I encodes trans-activator p40x and two other phosphoproteins. 302 74

A 67-nucleotide portion of the non-coding, 5'-leader sequence of tobacco mosaic virus RNA [defined as omega' (Gr. omega prime)] has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo. Chemically-synthesized omega', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII). Run-off in vitro transcripts, with or without a 5'-cap structure (G(5')ppp(5')G) and/or the omega' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600). In all cases, the presence of omega' increased the translational expression of both reporter genes, typically between 2- to 10-fold. Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv. Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of omega' as a potential translational enhancer of gene expression in vivo.
 ...
PMID:The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. 357 95

The plasmid gene cat-86 specifies chloramphenicol-inducible, chloramphenicol acetyltransferase in Bacillus subtilis. The inducible regulation is independent of the promoter that is used to activate cat-86 and is independent of the cat-86 coding sequence. We have proposed that the regulation of cat-86 results from the transcription of a pair of inverted-repeat sequences that immediately precede the coding sequence. These transcripts are predicted to sequester the cat-86 ribosome binding site in a stable RNA stem-loop which, in theory, should block the ribosome binding site from pairing with 16S rRNA. Inducible expression of cat-86 may therefore result in part from regulation of the translation of cat-86 mRNA. However, chloramphenicol-induction correlates with increased levels of cat-86 mRNA and the RNA stem-loop preceding the cat-86 coding sequence structurally resembles a rho-independent transcription terminator. We have therefore tested the inverted-repeats as a potential site of transcription termination. Transcription studies performed in vitro using SP6 RNA polymerase and in vivo by S1 mapping demonstrate that a substantial fraction of the potential cat-86 transcripts terminate at a site immediately 3' to the inverted-repeats. The results of the in vivo experiments suggest that the termination signal may be partially relieved by growth of cells in chloramphenicol.
 ...
PMID:A transcription termination signal immediately precedes the coding sequence for the chloramphenicol-inducible plasmid gene cat-86. 392

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.
 ...
PMID:Rapid mutagenesis and purification of phage RNA polymerases. 911 96

An Escherichia coli strain containing two plasmids was developed for in vivo isolation of the phage SP6 RNA polymerase mutants. It was developed to isolate mutants with increased proficiency of termination at the SP6 terminator and/or with reduced elongation processivity. Mutations were randomly introduced into an N-terminal third of the polymerase gene that was placed under a lac promoter in one plasmid. In the other plasmid, a promoter-lacking lacZ gene modified for reduced translation efficiency was placed downstream of a tandem pair of the SP6 terminator located downstream of an SP6 promoter-chloramphenicol acetyltransferase gene. Termination-up mutants were selected in vivo as they rendered LacZ activity level lower than the wild-type, without reducing chloramphenicol resistance substantially. Three such mutants (M15L, M15S, and D117G) were purified, and their termination efficiencies were measured in vitro at two different intrinsic termination signals in the E. coli rrnB terminator t1 that are different in requiring RNA hairpin formation. All three mutations enhanced termination efficiencies in vitro at the SP6 terminator and the upstream signal of rrnB t1, but reduced the efficiency at the downstream signal of it. All the mutations reduced elongation processivity, as the mutants produced much less amounts of large transcripts (2.1 kb) than the wild-type but the similar amounts of small transcripts (up to 670 nt). Thus, the mutations, all reducing elongation processivity of the polymerase, exhibited opposite effects on the two types of intrinsic termination signals, suggesting that the two mechanisms involve different interactions with the phage RNA polymerase.
 ...
PMID:Bacteriophage SP6 RNA polymerase mutants with altered termination efficiency and elongation processivity. 1089 13