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)

Transfection of foreign DNA into eukaryotic cells has become an important tool in molecular biology. Based on the results of previous studies of the core structure of human adenoviruses, we have developed a novel transfection method. The procedure involves the in vitro reconstitution of foreign DNA-of viral or other origins-with the major core protein VII of adenovirus type 2 (Ad2) or protamine from salmon sperm. Both proteins are rich in basic amino acids and appear to share structural features. The DNA-protein complexes are added directly to the medium of eukaryotic cells. The in vitro formation of specific DNA-protein complexes can be assessed by gel electrophoretic analyses. Bovine serum albumin does not enter into specific complexes with DNA. Transfection of DNA-protein VII or DNA-protamine complexes results in their rapid transport into the cell nuclei. About 2-4 hr after transfection, up to 40% of the DNA added to cell cultures in complexes can be found in the nucleus, as compared with less than 10% of the DNA when other transfection methods are applied or when naked DNA is added to cell cultures. DNAs transfected by the new method into mammalian or insect cells retain their characteristic restriction patterns at least 48 hr after transfection and are expressed efficiently. Supercoiled circular plasmid DNAs are converted to open circular or linear DNA. Expression has been measured both for transiently expressed genes (chloramphenicol acetyltransferase gene, Ad2 DNA in human HeLa cells) and for genes that have been integrated into the host genome and are expressed permanently, such as the gene for neomycin phosphotransferase in hamster BHK21 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A novel method for transfection and expression of reconstituted DNA-protein complexes in eukaryotic cells. 382 90

Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA deltaME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon deltaME/76 with a deletion of 76 nucleotides in the 3'UTR of deltaME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon deltaME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of deltaME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (deltaME/76CAT[+])- and minus (deltaME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental deltaME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of deltaME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the deltaME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system.
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PMID:Subgenomic replicons of the flavivirus Kunjin: construction and applications. 899 75

The induction of an efficient CD4(+) T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4(+) T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10(4) transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.
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PMID:Efficient class II major histocompatibility complex presentation of endogenously synthesized hepatitis C virus core protein by Epstein-Barr virus-transformed B-lymphoblastoid cell lines to CD4(+) T cells. 973 74

The effect of X-irradiation on production of MUC1 was studied with human colon carcinoma HT-29 cells. As evaluated by immunocytochemical staining, the percentages of MUC1-positive cells in cells at 4 days after 6 Gy irradiation and in unirradiated control cells were 52 +/- 3.5% (n = 6) and 26 +/- 2.8% (n = 6), respectively. Flow-cytometric analysis of living cells showed that MUC1 began to rise from day 1, reaching a plateau by day 4 after 6 Gy irradiation. Western blot analysis with monoclonal antibody MY.1E12 against glycosylated MUC1 (mature form) showed dose-dependent increases of two bands (500 and 390 kDa) corresponding to two polymorphic MUC1 alleles. Premature forms of MUC1 (350 and 240 kDa) were detectable with monoclonal antibody HMFG-2 only in irradiated cells, suggesting that new core protein synthesis had been induced. The transcriptional activity of the MUC1 gene was analyzed in terms of transient expression of MUC1-CAT reporter plasmids containing 5'-flanking sequences of the MUC1 gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The results of CAT assay indicate that enhanced expression of MUC1 in irradiated HT-29 cells was due to upregulation of MUC1 transcription, and required the upstream promoter.
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PMID:Increased expression after X-irradiation of MUC1 in cultured human colon carcinoma HT-29 cells. 1076 Jun 92