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 early gene which augments the expression of the delayed early/late 39K gene of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) was identified by functional mapping. Transient expression of the plasmid p39CAT, containing the bacterial chloramphenicol acetyltransferase coding sequences under the control of the promoter of the 39K protein, was observed in cells cotransfected with AcNPV DNA digested with several restriction endonucleases. However, when p39CAT was cotransfected with viral DNA digested with Bg/II restriction endonuclease, no CAT activity could be detected. To map the location of the Bg/II-sensitive sequences required for efficient expression of 39CAT, p39CAT and Bg/II-digested viral DNA were cotransfected with a PstI library of AcNPV DNA. The PstI-N fragment restored 39CAT activity. A major early 1.3-kb transcript from this fragment was mapped by S1 nuclease analysis. Transient assay experiments indicated that this major transcript of the PstI-N fragment was produced by an immediate early gene, named IE-N. The PstI-N fragment alone did not activate expression of p39CAT but was required when IE-1 was present in limiting quantities.
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PMID:Functional mapping of an AcNPV immediately early gene which augments expression of the IE-1 trans-activated 39K gene. 327 28

Mouse ornithine decarboxylase (ODC) genomic clones were isolated from a bacteriophage lambda genomic library representing mouse myeloma 653-1 cells which over-produce ODC due to amplification of an active ODC gene. Sequence analysis of the amplified ODC gene revealed that ODC mRNA is encoded by 12 exons, 10 of which (exons 3 to 12) code for the ODC protein. Exon 12 also corresponds to the 3' noncoding region of the two species of ODC mRNA which are formed by alternative utilization of two polyadenylation signals separated from each other by 422 nucleotides. The transcription initiation site was mapped by S1 nuclease protection and by primer extension analysis. The 5' flanking region is extremely rich in G + C and contains typical promoter motifs such as the TATA box and SP1 transcription factor binding sites. Joining the 5' flanking region to the Escherichia coli chloramphenicol acetyltransferase structural gene and its introduction into mouse cells resulted in the expression of a high level of chloramphenicol acetyltransferase activity. Comparing the sequence of the ODC gene to our previously published sequence of ODC cDNA revealed a disagreement between the sequences located 5' to the AvaI site and demonstrated that this region of our previously reported cDNA represents a cloning artifact. The portion of the correct 5' noncoding region encoded by exon 1 is extremely rich in G + C and includes potential secondary structures which may be involved in translational regulation of ODC mRNA.
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PMID:Isolation and characterization of the mouse ornithine decarboxylase gene. 337 2

The 5' end of the Chinese hamster ovary aprt gene was sequenced and transcription start sites were determined by both S1 nuclease protection and primer extension assays. Deletion mutants covering the same area were constructed, and adenine phosphoribosyltransferase (APRT) or chloramphenicol acetyltransferase (CAT) activity was measured by transient-expression assays. The aprt gene uses a single cluster of transcription start sites and lacks consensus sequences such as TATA and CCAAT, which are general components of eucaryotic promoters. The 5' deletion mutations of the promoter sequences demonstrated that (i) there is no decrease in either APRT activity or transcription extending to position -89 (relative to the main transcription start site); (ii) an additional 29-base-pair (bp) deletion decreases APRT activity and transcription twofold; and (iii) a deletion past the transcription start sites (P5' delta +27) abolishes both APRT activity and transcription, indicating that a 60-bp fragment immediately upstream of the main transcription start site is involved in basic transcription and a 29-bp fragment just upstream of the 60 bp-fragment stimulates transcription twofold. The 3' deletion mutations showed that a deletion of a 61-bp fragment in the 5' leader and coding sequence abolishes the efficient translation of an aprt-CAT gene transcript. In addition, there are two polyadenylation signals at the genomic 3' end, with the proximal one being sufficient for functional polyadenylation.
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PMID:Analysis of signals controlling expression of the Chinese hamster ovary aprt gene. 340 12

Vimentin is a growth-regulated gene whose mRNA levels increase severalfold after stimulation of quiescent cells. We have isolated and sequenced a genomic fragment of human DNA containing the vimentin 5'-flanking sequence and untranslated region. S1 nuclease analysis was used to determine the transcription initiation site. Deletion mutants of the promoter region were constructed, linked to a chloramphenicol acetyltransferase gene, and analyzed for transient expression by transfection into BALB/c 3T3 cells. These experiments revealed the presence in the human vimentin promoter region of a negative-regulatory element, flanked by positive elements. The most 5' of the positive elements is able to overcome the effects of the negative element. Analysis of these deletion constructs in stable cell lines confirmed the results of the transient assays. Using these stable cell lines, we can also demonstrate that the vimentin promoter region can confer platelet-derived growth factor inducibility to a linked chloramphenicol acetyltransferase gene and that the sequences required for this inducibility reside between positions -241 and +73.
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PMID:Functional analysis and growth factor regulation of the human vimentin promoter. 343 46

A series of recombinant plasmid vectors containing hepatitis B virus (HBV) DNA sequences was constructed to study the biosynthesis of the hepatitis B virus surface antigen (HBsAg) RNA and to locate transcriptional control elements involved in the regulation of the S and pre-S DNA sequences. We examined the transcription of the HBsAg gene in permanent cell lines that were developed by transfecting with recombinant vectors containing HBV sequences and the neomycin gene followed by G418 selection. We further defined the promoter activities upstream of and within the pre-S sequences using the assayable chloramphenicol acetyltransferase gene. Results obtained from S1 nuclease digestion and primer extension suggest that HBsAg transcripts are initiated at multiple sites in the pre-S region and from a site upstream of the pre-S region. Chloramphenicol acetyltransferase assays indicate that DNA sequences within and upstream of the pre-S region contain promoter activities and that the "TATA" sequence-containing promoter and the internal promoter show similar levels of activities in CV-1 cells and several other cell lines tested.
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PMID:Transcriptional control elements of hepatitis B surface antigen gene. 345 53

The expression of the human HSP70 gene is induced by a wide range of physiological stresses, including exposure to heat shock and heavy metals, or under nonstress conditions, such as in response to serum stimulation. We have previously demonstrated that in either case the regulated expression is at the primary level of transcription. To determine whether transcription is mediated through a single or multiple genetic elements, we have dissected the sequences upstream of the transcription start site of the human HSP70 gene by constructing chimeric genes retaining variable amounts of 5' flanking regions fused to the bacterial gene encoding chloramphenicol acetyltransferase. Transcription from the chimeric genes was determined by S1 nuclease analysis of separate stable transfectants. The sequences required for heat shock and cadmium induction lie between -107 and -68. Within this region is the sequence CTGGAATATTCCCG, which is identical in 12/14 positions with the heat shock element of Drosophila heat shock genes, and a separate sequence, CGNCCCGG, which is homologous to the core of the human metallothionein II metal-responsive element. The sequences required for serum-stimulated transcription are distinct from the heat shock element. The sequence CCAAT at -68 is required for high levels of correctly initiated transcripts, and a purine-rich sequence, GAAGGGAAAAG, at -58 is required for serum stimulation. The human HSP70 promoter contains at least two regulatory domains--a distal domain responsive to heat shock or cadmium and a proximal domain responsive to stimulation by serum.
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PMID:Human HSP70 promoter contains at least two distinct regulatory domains. 345 60

A plasmid containing 1.8 kilobase pairs of rat growth hormone (rGH) promoter and upstream flanking sequences fused to the bacterial gene for chloramphenicol acetyltransferase (CAT) was transiently introduced into pituitary, fibroblast, and kidney cell lines. Significant CAT activity was detectable only in the pituitary cell lines, demonstrating that this relatively large fragment directs strongly cell-type-specific expression. However, plasmids containing only 200-300 bases of rGH promoter and flanking sequences directed expression of CAT in all three cell types, suggesting that upstream sequences directly repress the activity of a minimal rGH promoter in nonpituitary cell types. S1 nuclease analysis showed that the RNA synthesis directed by one of the short rGH promoter fragments in fibroblasts initiated from the site used by the natural promoter in pituitary cells. Insertion of rGH upstream sequences in their natural orientation upstream of the mouse metallothionein I promoter caused a decrease in its activity in fibroblasts by a factor of 4, but there was a 2.5-fold increase in its activity in pituitary cells. Insertion of the rGH fragment upstream of the thymidine kinase promoter in either orientation lowered its activity in both fibroblasts and pituitary cells. Thus, the negatively acting rGH flanking sequences can act on a heterologous promoter and have at least some of the properties of positively acting enhancers.
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PMID:Repression mediates cell-type-specific expression of the rat growth hormone gene. 346 54

When Vero or murine cells were stably transfected with the human immunodeficiency virus (HIV) long terminal repeat (LTR) that directs the chloramphenicol acetyltransferase (CAT) gene (pU3R-III-CAT), expression was suppressed. Treatment with the nucleoside analog 5-azacytidine (5-azaC) restored CAT expression. S1 nuclease analysis and a nuclear run-on assay demonstrated that activation of the latent HIV LTR by 5-azacytidine occurred at the transcriptional level. Southern blot analysis demonstrated that this activation was due to the demethylation of cytosine residues in the LTR enhancer. Thus, the HIV LTR appears to be susceptible to transcriptional inactivation by methylation, a process that is proposed to play a modulatory role in viral latency.
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PMID:Methylation as a modulator of expression of human immunodeficiency virus. 346 17

We cloned and compared the sequence of a rearranged human T cell receptor (TCR) V alpha J alpha gene and its germline counterparts. The only difference in the coding region sequence was confined to the joining region where three nucleotides, TTG, unaccountable by either V alpha or J alpha sequence, were present. By nuclease S1 mapping we identified the mRNA start of the alpha chain 70 nucleotides upstream from the initiator ATG. A 600 bp fragment containing the sequences upstream to the ATG drives the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene. This promoter activity is T cell specific since it can be demonstrated in human T cells but not in B cells or HeLa cells. A 1.1 kb BamHI- HindIII fragment located 5' to the first exon of the C alpha gene was found to enhance transcription from either the heterologous SV40 promoter or the homologous TCR alpha chain promoter. This enhancement activity was independent of the location of the fragment with respect to CAT and was specific to lymphoid cells (either T or B cells) but cannot be demonstrated in HeLa cells.
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PMID:Promoter and enhancer elements in the rearranged alpha chain gene of the human T cell receptor. 350 68

Recombinant clones containing the promoter region of the human insulin receptor gene were isolated from genomic libraries derived from nondiabetic persons. A 1.5-kilobase pair fragment of the 5'-flanking region was sequenced. One transcriptional start site, located at 203 bases upstream from the start of translation was identified by nuclease S1 mapping and the primer extension experiment using the human insulin receptor mRNA. The bacterial chloramphenicol acetyltransferase assay revealed that a 573-base pair fragment immediately preceding the ATG has promoter activity and that the transcript initiates from the normal start site of the insulin receptor gene in the COS cells. The promoter region contains neither a "TATA box" nor a "CAAT box," has an extremely high G + C content, and contains seven central components of potential Sp 1 binding sites (GGGCGG or CCGCCC). These features are common to those found in the regulatory regions of a class of constitutively expressed "housekeeping" genes. A comparison between the promoter sequence of the human insulin receptor and those of other "housekeeping" genes revealed the presence of homologous sequences among these genes, in addition to the potential Sp 1 binding sites.
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PMID:Characterization of the promoter region of the human insulin receptor gene. Evidence for promoter activity. 368 Feb 48


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