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)

A very small plasmid vector system is described for construction and high-level production of C-terminal chloramphenicol acetyltransferase (CAT) fusion proteins in Escherichia coli. The only functional elements of the plasmid are a minimal region of the ColE1 origin of DNA replication and the Tn9 cat gene, both under control of a tac promoter. Since C-terminal fusion to CAT does not interfere with chloramphenicol (Cm) resistance, plasmids are maintained under Cm selection. Because of its small size (1392 bp), the system is especially convenient for building and expression of synthetic genes and gene fragments. This concept was utilized to generate a fusion with a synthetic gene encoding the multiple-epitope fragment from the rubella virus E1 membrane protein. Affinity-purified fusion proteins were obtained in mg amounts from 100-ml batches of culture fluid, and incorporated as a specific antigen in a rubella immunoglobulin G enzyme-linked immunosorbent assay.
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PMID:An Escherichia coli plasmid vector system for high-level production and purification of heterologous peptides fused to active chloramphenicol acetyltransferase. 768 30

Sequences at the 5' and 3' ends of the rubella virus (RV) genomic RNA can potentially form stable stem-loop (SL) structures that are postulated to be involved in virus replication. We have analyzed the function of these putative SL structures in RNA translation by constructing chimeric chloramphenicol acetyltransferase (CAT) RNAs, flanked either by both 5'- and 3'-terminal sequence domains from the RV genome or several deletion derivatives of the same sequences. After in vitro transcription of chimeric RNAs, the translational efficiencies of these RNAs were compared by the rabbit reticulocyte lysate translation system. For in vivo translation studies, the level of CAT activity was measured for chimeric RV/CAT RNAs expressed in transfected cells by the adenovirus major late promoter. Both in vivo and in vitro translation activities of the chimeric RNAs revealed that the presence of 5' and 3' SL sequences of RV RNA, in correct (+) orientation and context [5'(+)SL and 3'(+)SL, respectively] was necessary for efficient translation of chimeric RV/CAT RNAs. The presence of the RV 5'(+)SL sequence had the primary enhancing effect on translation. To identify host proteins which interact with the 5'(+)SL which may be involved in RV RNA translation, RNA gel-shift and UV cross-linking assays were employed. Two host proteins 59 and 52 kDa in size, present in cytosolic extracts from both uninfected and RV-infected cells, specifically interacted with the RV 5'(+)SL RNA. Direct binding comparisons between wild-type and mutant 5'(+)SL RNAs demonstrated that sequences in and around the bulge region of the terminal stem domain of this structure constituted a protein binding determinant. Human serum, qualified for anti-Ro/SS-A antigen specificity, immunoprecipitated 59- and 52-kDa protein-RNA complexes containing the RV 5'(+)SL RNA. However, poly- and monoclonal antisera raised against the recombinant 60- and 52-kDa Ro proteins failed to precipitate complexes containing the 5'(+)SL RNA. The identity of the proteins binding this RV cis-acting element remains to be determined; however, their role in RV translation is discussed.
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PMID:5' sequences of rubella virus RNA stimulate translation of chimeric RNAs and specifically interact with two host-encoded proteins. 769 67

Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Togaviridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors.
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PMID:Development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression. 1104 28

Proof of concept for a novel diagnostic assay for rubella virus (RUB) based on RUB replicons expressing reporter genes was demonstrated. RUB replicons have the structural protein coding region replaced with a reporter gene such as green fluorescent protein or chloramphenicol acetyltransferase. Previously, it was shown that a replicon construct with a specific in-frame deletion in the nonstructural protein coding region (NotI, approximately nucleotides 1500 to 2100 of the genome) failed to replicate and express the reporter gene unless rescued by a coinfecting wild-type helper RUB (W.-P. Tzeng et al., Virology 289:63-73, 2001). In the present study, it was found that rescue of reporter gene expression by NotI replicons occurred when coinfection was done with clinical specimens containing RUB, indicating that this system could be the basis for a diagnostic assay. The assay was sensitive, using laboratory RUB strains and as low a dose as one plaque-forming unit. The assay was specific in that it was positive for RUB strains of both genotypes and was negative for a panel of human viruses. It was also possible to genetically sequence the RUB present in positive clinical specimens detected in the assay for genotypic strain determination.
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PMID:Novel replicon-based reporter gene assay for detection of rubella virus in clinical specimens. 1569 95