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)

An in vitro RNA synthesis system was established in which the influenza virus virion (minus-sense) RNA was made from the synthetic plus-sense RNA (cRNA) template by the purified viral polymerase complex. The cRNA promoter was studied by mutational analysis using the in vitro system, and on the basis of these experiments, the first 11 nucleotides of the 3' noncoding sequence were found to contain the minimum promoter required for virion RNA synthesis. The addition of extra nucleotides at the 3' end decreased the promoter activity of the templates, indicating that the viral polymerase does not recognize an internal promoter efficiently. The wild-type and mutated RNA templates were also tested in vivo by using the ribonucleoprotein transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3'-terminal sequence significantly decreased or abolished chloramphenicol acetyltransferase (CAT) expression. These results suggest that the cRNA promoter overlaps other essential cis elements required for chloramphenicol acetyltransferase expression in vivo.
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PMID:Mutational analysis of the promoter required for influenza virus virion RNA synthesis. 160 47

A system for the expression of a foreign gene derived from negative polarity RNA was developed using influenza virus, a negative-stranded RNA virus. From cDNA for the influenza virus RNA genome segment 8, the region coding for the nonstructural protein was deleted and replaced by the chloramphenicol acetyltransferase (CAT) gene. The resulting DNA sequence was placed under the control of the promoter of T7 RNA polymerase such that the antisense RNA to CAT mRNA was produced when transcribed by T7 RNA polymerase. Transfection of HeLa cells with this antisense CAT RNA in the presence of the helper ribonucleoprotein cores led to no significant production of the CAT. In contrast, when the RNA was covered with purified nucleoprotein prior to transfection, the CAT gene was efficiently expressed. This indicated that the viral RNA polymerase transcribed the RNA transfected as the RNA-nucleoprotein complexes. In addition, this system was used for analysis of the cis-acting region in transcription and the promoter structure of the viral RNA genome.
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PMID:In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA. 205 14

The ribonucleocapsids of many plant viruses are extremely stable. The protein coat protects the RNA genome against degradation during the accumulation and spread of progeny virions. Chimeric single-stranded RNA molecules were transcribed in vitro from recombinant plasmids and later encapsidated, in vitro, into ribonucleoprotein particles (pseudoviruses) 60 nanometers long that resembled tobacco mosaic virus. Transcripts encoding an assayable enzyme, chloramphenicol acetyltransferase (CAT), were packaged into pseudovirus particles to assess the utility of this single-stranded RNA delivery system in a wide range of cell types. In all cases, packaged CAT messenger RNA was uncoated and transiently expressed. Significantly higher levels of CAT activity were detected with packaged than with naked CAT messenger RNA after inoculation of plant protoplasts in the presence of polyethylene glycol or abrasive inoculation of intact leaf surfaces. Structural events that lead to the uncoating and expression of CAT messenger RNA showed no cell specificity. This observation may support the view that the comparatively restricted host range of a true plant virus results from events that occur later during the infection cycle.
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PMID:In vivo uncoating and efficient expression of foreign mRNAs packaged in TMV-like particles. 347 50

The influenza virus NS1 protein was shown to stimulate translation of the M1 protein. M-CAT RNA, which contains the chloramphenicol acetyltransferase (CAT) reporter gene and the terminal noncoding sequence of segment 7 (coding for the M1 and M2 proteins), was ribonucleoprotein transfected into clone 76 cells expressing the influenza virus RNA polymerase and NP proteins required for the transcription and replication of influenza virus ribonucleoproteins. When the cells were superinfected with a recombinant vaccinia virus which expresses the NS1 protein, CAT expression from the M-CAT RNA was significantly stimulated but transcription was not altered. The expression of NS-CAT RNA, which contains noncoding sequences of segment 8 (coding for the NS1 and NS2 proteins), was not altered by the NS1 protein. Site-directed mutagenesis showed that the sequence GGUAGAUA upstream of the initiation codon on segment 7 was required for stimulation.
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PMID:Influenza virus NS1 protein stimulates translation of the M1 protein. 750 95

The human insulin-like-growth-factor-2 (IGF-2) gene generates mRNAs with four different leader sequences, but with identical coding and trailing regions. Previous research has revealed that the leader-2-containing and leader-4-containing mRNAs are completely polysomal, whereas mRNAs possessing leader-3 are predominantly present in the untranslated free messenger ribonucleoprotein particle (mRNP), both in cell lines and in foetal liver tissue. To investigate the influence of the IGF-2 leader sequences on expression of the gene, IGF-2 leader-luciferase and leader-chloramphenicol acetyltransferase fusion constructs were transfected transiently into different cell lines. In these experiments, the levels of expression obtained by constructs with leader-1, leader-2 and leader-4 were very similar, both at the level of mRNA and protein. Leader-3, however, strongly repressed the expression of the fusion mRNA via an unknown mechanism. This repression appeared to be confined to nucleotides at positions 328-906 of the leader sequence. The remaining 5' part of the leader sequence was efficient both in RNA expression and in translation, but the 3' part of the leader (nucleotides 906-1180) again moderately repressed luciferase expression, possibly due to endonucleolytic cleavage in this region of the RNA. To evaluate the effect of the IGF-2 leaders on in vitro translation, leader-chloramphenicol acetyltransferase fusion mRNAs were synthesized and translated in reticulocyte lysates. Compared to a chloramphenicol acetyltransferase control RNA, leader-1-chloramphenicol acetyltransferase mRNA translated over 20-fold less efficiently, whereas leader-2 repressed translation of its chloramphenicol acetyltransferase mRNA moderately (3-5 fold). Despite a general improvement of the translation efficiency upon translation in HeLa lysate, these discrepancies with the transfection data persisted. Translation of leader-3-containing mRNAs in reticulocyte lysates was barely detectable. The whole 5' region of leader-3, up to nucleotide 614, could be shown to be repressive. Only leader-4 directed translation of the chloramphenicol acetyltransferase open reading frame efficiently. As with leader-1 and leader-2, this L4-chloramphenicol acetyltransferase mRNA translated in a cap-dependent manner under the conditions of our experiments; translation of this mRNA was relatively resistant to addition of cap analogue. We conclude that all four IGF-2 leader sequences differ in their translational properties. This makes it likely that changes in the translational machinery will affect the expression of the various IGF-2 mRNAs differentially.
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PMID:Influence of the four leader sequences of the human insulin-like-growth-factor-2 mRNAs on the expression of reporter genes. 781 58

An in vivo system in which expression of a synthetic influenza virus-like chloramphenicol acetyltransferase (CAT) RNA is driven by influenza virus proteins synthesized from cloned cDNAs has been developed. Expression of the four influenza virus core proteins (nucleoprotein, PA, PB1 and PB2) was performed by transfection of four pGEM recombinant plasmids, each containing one of the four viral genes, into cell cultures previously infected with a vaccinia virus recombinant encoding the T7 RNA polymerase (vTF7-3). When a naked negative-sense influenza virus-like CAT RNA was transfected into cells expressing the four influenza virus proteins, CAT activity was detected in the cell extracts, demonstrating that the expressed proteins had RNA-synthesizing activity. In this system, CAT RNA templates containing additional nucleotides at the 3' end were also expressed, resulting in CAT activity. This showed that the influenza virus polymerase can recognize its promoter when located internally on an RNA template. In influenza virus-infected cells however, CAT activity was detected only when the CAT RNA contained the viral promoter at the exact 3' end and was transfected as in vitro assembled ribonucleoprotein. These results are discussed in terms of the different requirements of the two helper systems for expression of an exogenously added RNA.
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PMID:Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system. 804 17

The functionality of the influenza virus polymerase subunits and the nucleoprotein expressed from simian virus 40 (SV40) recombinants has been tested by their ability to direct the in vivo expression of influenza virus-like RNAs. These RNAs, which contained either the chloramphenicol acetyltransferase (CAT) or haemagglutinin (HA) genes, were synthesized and reconstituted in vitro into viral ribonucleoproteins with a polymerase/nucleoprotein mixture purified from influenza virus-infected cells. Only the coinfection with SV40 recombinant viruses expressing the three polymerase subunits and the nucleoprotein allowed the expression of the transfecting CAT or HA RNAs, confirming that this set of viral genes is the minimal requirement for viral gene expression. Unexpectedly, transfection of the corresponding naked RNAs into SV40 recombinant-infected cells was as effective in directing the synthesis of CAT or HA proteins as the standard reconstituted ribonucleoprotein transfection. These results may be important for the genetic analysis of trans-acting factors involved in influenza virus transcription and replication and may open the way to rescuing influenza viruses in the absence of a helper virus.
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PMID:Influenza virus naked RNA can be expressed upon transfection into cells co-expressing the three subunits of the polymerase and the nucleoprotein from simian virus 40 recombinant viruses. 838 87

Recently, the vesicular stomatitis virus matrix (M) protein has been shown to be capable of inhibition of host cell-directed transcription in the absence of other viral components (B. L. Black and D. S. Lyles, J. Virol. 66:4058-4064, 1992). M protein is a major structural protein that is known to play a critical role in virus assembly by binding the helical ribonucleoprotein core of the virus to the cytoplasmic surface of the cell plasma membrane during budding. In this study, two M protein mutants were tested to determine whether the inhibition of host transcription by M protein is an indirect effect of its function in virus assembly or whether it represents an independent function of M protein. The mutant M protein of the conditionally temperature-sensitive (ts) vesicular stomatitis virus mutant, tsO82, was found to be defective in its ability to inhibit host-directed gene expression, as shown by its inability to inhibit expression of a cotransfected target gene encoding chloramphenicol acetyltransferase. The ability of the tsO82 M protein to function in virus assembly was similar to that of wild-type M protein, as shown by its ability to complement the group III ts M protein mutant, tsO23. Another mutant, MN1, which lacks amino acids 4 to 21 of M protein demonstrated that the abilities of M protein to inhibit chloramphenicol acetyltransferase gene expression and to localize to the nucleus were unaffected by deletion of this lysine-rich amino-terminal region but that the ability to function in virus assembly was ablated. Thus, the two M protein mutants examined in this study exhibited complementary phenotypes: tsO82 M protein functioned in virus assembly but was defective in inhibition of host-directed gene expression, while MN1 M protein functioned in inhibiting gene expression but was unable to function in virus assembly. These data demonstrate that the role of M protein in inhibition of host transcription can be separated genetically from its role in virus assembly.
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PMID:The role of vesicular stomatitis virus matrix protein in inhibition of host-directed gene expression is genetically separable from its function in virus assembly. 839 15

The large (L) protein of nonsegmented negative-strand RNA viruses is the multifunctional catalytic component of the viral ribonucleoprotein (RNP) complex. To address the role of conserved rabies virus (RV) L protein sequences predicted to be involved in RNA polymerase activity, a reverse genetics approach was applied that allows intracellular reconstitution of transcriptionally active RV RNPs from plasmid-encoded proteins. Artificial RV model genomes encoding bacterial chloramphenicol acetyltransferase or firefly luciferase was used to determine the polymerase activity of a series of 23 RV L proteins mutated in the highly conserved C motif of the proposed polymerase module. All constructs with mutations of the GDN core sequence of motif C, which is proposed to be a variant of the catalytical XDD residues of RNA polymerase and reverse transcriptases, failed to express the reporter genes. In addition, the identity of the upstream residues AQ was crucial for maintenance of polymerase activity. Several conservative and nonconservative mutations introduced into the three amino acids QVL located downstream of the GDN core resulted in reduced polymerase activities and expression of luciferase in the range 0.4 to 92% compared to the parental L protein.
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PMID:Polymerase activity of in vitro mutated rabies virus L protein. 855 54

A reverse genetics approach was applied to generate a chimeric nonsegmented negative strand RNA virus, rabies virus (RV) of the Rhabdoviridae family, that expresses a foreign protein. DNA constructs containing the entire open reading frame of the bacterial chloramphenicol acetyltransferase (CAT) gene and an upstream RV cistron border sequence were inserted either into the nontranslated pseudogene region of a full-length cDNA copy of the RV genome or exchanged with the pseudogene region. After intracellular T7 RNA polymerase-driven expression of full-length antigenome RNA transcripts and RV nucleoprotein, phosphoprotein and polymerase from transfected plasmids, RVs transcribing novel monocistronic mRNAs and expressing CAT at high levels, were recovered. The chimeric viruses possessed the growth characteristics of standard RV and were genetically stable upon serial cell culture passages. CAT activity was still observed in cell cultures infected with viruses passaged for more than 25 times. Based on the unprecedented stability of the chimeric RNA genomes, which is most likely due to the structure of the rhabdoviral ribonucleoprotein complex, we predict the successful future use of recombinant rhabdovirus vectors for displaying foreign antigens or delivering therapeutic genes.
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PMID:Highly stable expression of a foreign gene from rabies virus vectors. 869 89


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