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)

High levels of expression of cloned genes have been obtained in mammalian cells by using poxvirus-derived insertion/expression vectors. These vectors employ the cis-acting element (CAE I) that directs the transcription of one of the most strongly expressed genes of cowpox virus. This gene (the 160K gene) encodes the 160-kDa protein that is the major component of the A-type cytoplasmic inclusions. Its counterpart in vaccinia virus (VV) is the 94K gene contained in the HindIII A fragment of the viral DNA. Two insertion vectors have been constructed; each is designed to allow cloned genes to be placed immediately downstream of a modified version of CAE I within a poxvirus genome. One vector, p1200, enables the CAE I-cloned-gene constructs to be inserted into the thymidine kinase gene of VV. This vector was used to create a VV recombinant that directed expression of the chloramphenicol acetyltransferase (CAT) gene. The other vector, p2101, enables the CAE I-cloned-gene constructs to be inserted into the VV 94K gene. The prototype of this vector was used to create a VV recombinant that directed expression of a hybrid CAT-lacZ gene. Infection of cultured human cells with these recombinants led to high levels of synthesis of either the CAT gene product or the CAT-lacZ gene product. Each of these proteins was produced in quantities that were easily detected by Coomassie blue staining of total cell proteins resolved by polyacrylamide gel electrophoresis. We estimate that these vectors are capable of directing the synthesis of milligram amounts of gene product per 10(9) mammalian cells.
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PMID:A poxvirus-derived vector that directs high levels of expression of cloned genes in mammalian cells. 284 5

Vaccina virus (VV) and cowpox virus (CPV) differ in their abilities to replicate in Chinese hamster ovary (CHO) cells because VV has a disrupted host range (hr) gene. To facilitate an examination of the molecular events associated with abortive infection of CHO cells with VV, we constructed two sets of recombinant viruses that contain a viral early promoter regulating the cat gene encoding chloramphenicol acetyltransferase and viral intermediate or late promoters regulating the lacZ gene encoding beta-galactosidase. The first set has the disrupted hr gene and the second set has the intact CPV homolog, allowing replication in CHO cells. Reporter chloramphenicol acetyltransferase and beta-galactosidase assays demonstrated that early gene expression was unperturbed, whereas intermediate and late gene expression were severely inhibited under abortive conditions. Metabolic labeling studies confirmed the absence of viral late protein synthesis. The accumulation of viral DNA under abortive conditions was consistent with the synthesis of viral early proteins and established that inhibition of late protein synthesis was not primarily due to a replicative block. Analysis of steady state levels of viral mRNAs revealed substantial quantities of early and intermediate species but only very small amounts of late mRNAs under nonpermissive conditions. Despite the presence of viral intermediate mRNAs, the corresponding intermediate proteins, which function as late transcription factors, were not detected by immunoprecipitation of lysates from metabolically labeled infected CHO cells. Furthermore, when expression of lacZ was regulated by an intermediate promoter, no beta-galactosidase was detected even though lacZ transcripts were present. Thus, the abortive phenotype in CHO cells can be explained by a block to translation of intermediate mRNAs which prevents the synthesis of late transcription factors.
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PMID:Restriction of vaccinia virus replication in CHO cells occurs at the stage of viral intermediate protein synthesis. 785 9

The vaccinia virus/bacteriophage T7 expression system was adapted to Chinese hamster ovary (CHO) cells. Vaccinia virus undergoes abortive infection in CHO cells, which is characterized by a sharp reduction in protein synthesis at the stage of viral intermediate gene expression. We determined that expression of a T7 promoter-regulated chloramphenicol acetyltransferase gene was at least 20 times more efficient in permissive BS-C-1 than in CHO cells. The encephalomyocarditis virus 5'-untranslated region, which confers cap-independent translatability to mRNA, stimulated recombinant protein synthesis by 10-fold in both cell lines, maintaining the advantage of the BS-C-1 cells over CHO cells. Since the cowpox virus hr gene overcomes vaccinia virus host range restriction in CHO cells, we constructed a recombinant virus that carries an intact hr gene in addition to the T7 RNA polymerase gene. With this virus, synthesis of T7 RNA polymerase was enhanced and production of a recombinant protein occurred in CHO cells at the level observed in permissive cell lines. Extension of the vaccinia virus/bacteriophage T7 expression system to CHO cells should be of wide interest, as these cells have advantages for preparation of recombinant proteins in research and biotechnology.
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PMID:Recombinant protein synthesis in Chinese hamster ovary cells using a vaccinia virus/bacteriophage T7 hybrid expression system. 866 85