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)

To determine the block(s) to spleen necrosis virus (SNV) replication in mouse cells, we studied the expression of a dominant selectable marker, neo, or a gene whose product is easily assayed, the chloramphenicol acetyltransferase (cat) gene, in SNV-derived and murine leukemia virus-derived vectors. Using transient (CAT) and stable (Neor phenotype) transfection assays, we showed that the SNV promoter was used in mouse cells only when the 3' SNV long terminal repeat (LTR) was absent. Infection of mouse cells with recombinant SNV viruses was 1% as efficient as infection of permissive dog (D17) cells. The SNV proviruses in mouse cells appeared normal by Southern blot analysis, indicating that their integration probably occurred by normal mechanisms. S1 nuclease analyses of Neor mouse cell clones, each harboring a single recombinant SNV provirus, showed that the selected (internal) promoter was active, but that the 5' SNV LTR promoter was not. However, in the rare (less than 10(-6)) Neor colonies in which expression of the 5' LTR was selected, both promoters were active. Thus, the block to SNV infection of mouse cells is at least at two levels; one is a 100-fold-decreased efficiency at some step(s) up to and including integration, and the other is at transcription.
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PMID:Transcription from a spleen necrosis virus 5' long terminal repeat is suppressed in mouse cells. 244 16

By cloning MboI fragments in the promoter selection vector pGKV210, which replicates in Streptococcus lactis, Bacillus subtilis, and Escherichia coli and carries a promoterless chloramphenicol acetyltransferase gene, we obtained a number of fragments endowed with promoter activity, partly by direct selection for chloramphenicol resistance in S. lactis IL1403 and partly by selection in B. subtilis. Five fragments were sequenced, and the promoters were mapped with S1 nuclease. The promoters agreed with the E. coli promoter consensus and the B. subtilis vegetative sigma 43 promoter consensus. The promoters were preceded by an A + T-rich region (ranging from 64 to 78% A + T). S1 nuclease mapping data showed that the transcriptional start point in three of the fragments was at a TAG sequence 5 to 9 nucleotides downstream from the promoter. Three fragments carried an open reading frame preceded by a ribosome-binding site which can be recognized by E. coli, B. subtilis, and S. lactis ribosomes.
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PMID:Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. 244 29

The transcriptional activity of five intracisternal A-particle (IAP) long terminal repeats (LTRs) in mouse embryonal carcinoma PCC3-A/1 cells and in Ltk- cells was determined. We tested the promoter activity of the LTRs by coupling them to the reporter gene chloramphenicol acetyltransferase (CAT) or guanosine-xanthine phosphoribosyltransferase (gpt). Each LTR was tested for promoter function in both the sense (5' to 3') and antisense (3' to 5') orientation preceding the reporter gene. The transcriptional activity of individual IAP gene LTRs varied considerably, and the LTR from IAP81 possessed promoter activity in both directions. The bidirectional activity of the IAP81 LTR confirmed by monitoring Ecogpt expression in stably transfected Ltk- cells, with the initiation sites for sense and antisense transcription being localized to within the IAP81 LTR by S1 nuclease mapping. Deletions of LTR81 show that for normal 5'-to-3' gene transcription (sense direction), the 3'U3/R region determines the basal level of transcription, whereas sequences within the 5'U3 region enhance transcription four- to fivefold. Deletion mapping for antisense transcription indicates that a 64-base-pair region (nucleotides 47 to 110) within the U3 region is essential for activity. These data indicate that the U3 region contains all the regulatory elements for bidirectional transcription in IAP LTRs.
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PMID:Functional analysis of the long terminal repeats of intracisternal A-particle genes: sequences within the U3 region determine both the efficiency and direction of promoter activity. 245 71

We located and characterized transcription terminators in the E1a and E1b genes by transferring the 3' fragments of the genes into the vector pSCAT10 [Sato et al., Mol. Cell. Biol. 6 (1986) 1032-1043] at a site located immediately downstream from the cat gene (coding for chloramphenicol acetyltransferase; CAT) and upstream from the simian virus 40 polyadenylation region. Multiple terminators were located downstream from the E1b gene, but not in the 3' region of the E1a gene. Fine analysis of these terminators by the CAT assay method and S1 nuclease mapping of in vivo transcripts indicated possible involvement of a G-rich sequence in transcription termination of the E1 region. These terminators were repeated tandemly and used by both the E1a and the E1b genes in a orientation-dependent manner.
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PMID:Multiple transcription terminators in E1a and E1b genes of adenovirus type 5. 246 6

Thrombospondin (TSP) is an extracellular matrix glycoprotein whose synthesis and secretion by mesenchymal cells is regulated at the level of gene transcription by platelet-derived growth factor. To examine the transcriptional regulation of the TSP gene at the molecular level, a genomic clone containing the human TSP promoter and flanking sequence was isolated and characterized. A 3.8-kilobase pair (kb) DNA fragment containing the first three exons, the first two introns, and 2.2 kb of 5'-flanking region was sequenced, and the site of transcription initiation was determined by both primer extension and S1 nuclease mapping. Consensus sequences for several potential regulatory elements were found in the 5'-flanking sequence, including a TATA box consensus sequence, TTTAAAA, located 24 base pairs upstream from the transcription start site. A chimeric gene was constructed containing the first intron, the first exon, and 2.0 kb of 5'-flanking sequence of the TSP gene fused to the promoterless gene for chloramphenicol acetyltransferase. When transfected into COS-1 or NIH3T3 cells this gene construct was transcribed, indicating the presence of a functional promoter in the TSP sequence. Transient transfection studies using deletion mutants of this TSP-chloramphenicol acetyltransferase construct were performed to locate cis-acting regulatory sequences. The deletion of flanking sequence 5' to position -234 had little or no effect on transcriptional activity, whereas deletion of 5'-flanking sequence extending further in the 3' direction resulted in the gradual loss of transcriptional activity. The removal of the first intron resulted in a 4-fold decrease in transcript levels, indicating the presence of a cis-acting positive element(s) in the first intron of the human TSP gene. This element(s) was further localized to the region between position +576 and position +727.
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PMID:Characterization of the promoter region of the human thrombospondin gene. DNA sequences within the first intron increase transcription. 254 87

The 5' flanking region of the mouse insulin proreceptor gene was isolated, and the 5' boundary of the minimal promoter was mapped. Genomic clones encompassing greater than 30 kilobases of the gene contain the promoter and exons 1 and 2 interrupted by an approximately 20-kilobase intron at the codon for amino acid 7 of the alpha subunit. The nucleotide sequence of a 1.3-kilobase fragment containing 766 base pairs of the 5' flanking region and the entire first exon was determined. Two major transcription start sites were mapped by S1 nuclease analysis to sites located 469 and 424 nucleotides upstream from the initiation codon for translation. The 5' terminus of an insulin proreceptor cDNA, isolated from a mouse 3T3-L1 adipocyte cDNA library, corresponds to the 3'-most major start site of transcription. The 5' deletion mutants of the 5' flanking region of the proreceptor gene, linked upstream of the bacterial chloramphenicol acetyltransferase reporter gene, were transfected into 3T3-L1 preadipocytes and assayed for promoter activity. The 5' boundary of the minimal promoter, which directs unexpectedly high levels of reporter gene expression, maps to a region 22 base pairs upstream from the 3'-most major transcription start site.
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PMID:Characterization of the mouse insulin receptor gene promoter. 260 74

We report the isolation and complete sequence of the gene encoding the rabbit erythroid-cell-specific 15-lipoxygenase (RBC 15-LOX), containing 14 exons spanning 8.0 kb. The transcription start point was mapped by S1 nuclease-protection experiments and comparison with the sequence of the RBC 15-LOX mRNA, as defined previously by primer extension experiments. The promoter contains a TATA-like motif, but no CCAAT motif in the canonical position, and lies within a 'CpG-rich island'. Functional analysis of the immediate 5'-flanking DNA by transfection experiments shows that a 150 nucleotide (nt) 5' fragment linked to the chloramphenicol acetyltransferase gene acts as a functional promoter in both erythroid and nonerythroid cell lines and responds in an erythroid-specific manner to the enhancer from the Friend murine leukaemia virus long terminal repeat, whereas a 40-nt fragment is inactive. Intron 7 contains eight copies of a 54-nt repeat containing a region with homology to the simian virus 40/immunoglobulin gene enhancers.
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PMID:The promoter structure and complete sequence of the gene encoding the rabbit erythroid cell-specific 15-lipoxygenase. 261 16

Two distinct regions of the transforming growth factor (TGF)-beta 1 promoter are responsive to autoregulation. Sequences located between nucleotides -454 to -323 and between the two major transcriptional start sites have positive regulatory activities and are induced by TGF-beta 1 in A-549 cells. The chloramphenicol acetyltransferase activity of the upstream human TGF-beta 1 promoter-chloramphenicol acetyltransferase gene is increased 8- to 10-fold by treatment of cells with TGF-beta 1, whereas that of the second promoter is increased approximately 3- to 4-fold. Using an S1 nuclease protection assay of chloramphenicol acetyl-transferase mRNA, we found that the steady-state expression of chloramphenicol acetyltransferase mRNA also is markedly increased. Seven distinct factors present in nuclear extracts from A-549 cells interact with the sequences between -454 and -323, strongly supporting the involvement of sequence-specific transcription factors in the transcriptional autoactivation of the human TGF-beta 1 gene.
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PMID:Promoter sequences of the human transforming growth factor-beta 1 gene responsive to transforming growth factor-beta 1 autoinduction. 270 52

Regulation of albumin gene expression is believed to be mediated by multiple nuclear factors that interact with cis-acting DNA sequences within the first 160 base pairs (bp) of the promoter. The minimal promoter sequence required to generate tissue-specific expression has not been clearly defined. We have constructed a series of transient expression vectors containing progressive deletions of the mouse albumin gene 5'-flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and include the Moloney murine leukemia viral (Mo-MuLV) enhancer. Promoter activity was determined in mouse hepatoma and fibroblast cell lines by chloramphenicol acetyltransferase and S1 nuclease analyses. All constructions were compared with -623 Albcat-Mo-MuLV which contains all the sequence homology between the rat and mouse promoters. Low levels of expression were observed with -60 Albcat-Mo-MuLV (10%) in hepatoma but not fibroblast cells. Addition of promoter sequence to -208 bp progressively increased activity to 190% in the hepatoma cells, while -308 and -1612 Albcat-Mo-MuLV had activity similar to the -623 Albcat-Mo-MuLV level, and -3000 Albcat-Mo-MuLV showed a 2-fold reduction in transcriptional activity. The inclusion of promoter sequences upstream of -60 generated low levels of expression in the fibroblasts. We also show that factors from mouse liver nuclear extracts protect at least five regions of the albumin promoter upstream of -160. Our results indicate that tissue specificity is established within the proximal promoter region and that additional cis-acting elements that may have a functional role in the efficiency of albumin gene expression are located upstream of -160 bp.
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PMID:Cell-specific expression of mouse albumin promoter. Evidence for cell-specific DNA elements within the proximal promoter region and cis-acting DNA elements upstream of -160. 272 22

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.
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PMID:Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression. 282 8


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