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)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to interact with a cytosolic receptor and, in turn, activate transcription of the mouse P1(450) gene. Various lengths of DNA upstream of the P1(450) gene were inserted into the pSV0-cat expression vector, with and without addition of the Harvey murine sarcoma virus (Ha-MSV) 72-bp repeat enhancer element. The constructs were cotransfected with pSV2-neo into mouse hepatoma wild-type cells and two variant cell lines. One variant is believed to result from a mutation in the P1(450) structural gene and expresses high levels of P1(450) mRNA constitutively; the other variant has a defect in nuclear translocation of the inducer-receptor complex. After selection in G418, the cells were treated with control medium, TCDD, cycloheximide, or TCDD plus cycloheximide and then assayed for chloramphenicol acetyltransferase (CAT) activity. The data are consistent with the presence of several functional regions within the upstream sequence: a promoter region, a region that is negatively autoregulated, possible repressor-binding and inducer-receptor complex-binding sites, and an upstream activation element that is required for transcriptional activation by TCDD. The Ha-MSV enhancer can substitute for this upstream activation element.
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PMID:Autoregulation plus upstream positive and negative control regions associated with transcriptional activation of the mouse P1(450) gene. 299 46

It has recently been shown that hepatitis B virus (HBV) contains a transcriptional enhancer element. In order to determine whether this enhancer responds to glucocorticoids, a series of derivatives of plasmid pA10CAT2 was constructed containing the HBV enhancer and variable lengths of further upstream sequences. Transient expression of chloramphenicol acetyltransferase (CAT) was determined after introduction of these plasmids into PLC/PRF/5, Hep 3B, Hep G2, HeLa, and mouse L cells. Highest CAT activity was noted in the human hepatocellular carcinoma line PLC/PRF/5, which contains integrated HBV DNA sequences. Dexamethasone augmented CAT expression in all cell lines tested with 40% of maximal induction at 10 nM and maximum stimulation (3- to 8-fold) at 1 microM dexamethasone. Dexamethasone augmentation of CAT expression was observed only when constructs contained HBV DNA sequences residing upstream to map position 735 from the EcoRI site. This indicates that the glucocorticoid-responsive region is distinct from the previously defined HBV enhancer sequence located at map position 1080-1234. These studies suggest that HBV DNA contains a glucocorticoid-responsive element, which may mediate expression of HBV genes in infected mammalian cells.
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PMID:Hepatitis B virus DNA contains a glucocorticoid-responsive element. 300 59

Ornithine transcarbamylase (OTCase) is a mitochondrial matrix enzyme that catalyzes the 2nd step in the mammalian urea cycle. The gene encoding OTCase is located on the X chromosome and expression of OTCase is limited almost exclusively to hepatocytes. We have characterized a lambda phage recombinant, isolated from a mouse genomic library, that spans the first two exons of the mouse OTCase gene. Nuclease S1 mapping and primer extension analysis of this clone allowed us to determine that the transcription start site is 136 base pairs (bp) upstream from the translation initiation codon. Two TATA-like sequences were found 25 and 153 bp from the transcription initiation point. An 800-bp fragment containing the 5' flanking region of the OTCase gene was fused upstream to the coding sequence of the chloramphenicol acetyltransferase gene to assay promoter activity. This plasmid was introduced into mouse fibroblast NIH 3T3 cells and human hepatoma Hep G2 cells by the calcium phosphate co-precipitation method. After DNA transfection chloramphenicol acetyltransferase activity was observed only in Hep G2 cells. We conclude that this 800-bp fragment contains sufficient information to control OTCase gene expression in a tissue-specific manner, probably by interacting with trans-acting factor(s) which are not present in the other cell line.
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PMID:The 5' flanking region of the ornithine transcarbamylase gene contains DNA sequences regulating tissue-specific expression. 301 88

In mouse hepatoma Hepa-1 cells, polycyclic aromatic compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activate transcription of the mouse P(1)450 gene via trans-acting regulatory factors that include the TCDD X receptor complex. The positive control element in the P(1)450 5'-flanking region was examined in control and TCDD-treated Hepa-1 stable transformants that had been transfected with either of two expression vectors containing the chloramphenicol acetyltransferase (CAT) gene: pA10-cat, which has the simian virus 40 (SV40) early core promoter (without enhancers) immediately upstream from the CAT gene; and pSV0-cat, which has no promoter or enhancer. When the 1-kb DNA fragment from -1,647 to -611 upstream from the P(1)450 gene is inserted in either orientation--immediately upstream or almost 2 kb further upstream--from the SV40 promoter in pA10-cat, there is enhancement of CAT activity that can be further induced three- to fourfold by TCDD. When the same experiment is carried out with the -1,247 to -823 fragment or the -1,051 to -823 fragment, but not the -1,247 to -1,052 fragment, TCDD responsiveness is lost, or at least masked, because of a large increase in constitutive CAT activity. pSV0-cat mutants containing internal deletions in the upstream flanking sequences of P(1)450 were constructed. A region of 300 bases (-1,218 to -918) is shown to be required for TCDD responsiveness, and one TCDD-inducible element can be dissociated from an enhancer of constitutive gene expression, whereas one or more other TCDD-inducible elements cannot.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dioxin-inducible enhancer region upstream from the mouse P(1)450 gene and interaction with a heterologous SV40 promoter. 302

The transcriptional enhancer sequence has recently been demonstrated in the hepatitis B viral genome. This enhancer sequence has also been shown to increase the activity of HBcAg gene promoter. Using the chloramphenicol acetyltransferase gene expression system, we demonstrated that the intrinsic promoter activity of HBsAg gene promoter was stronger than that of HBcAg gene promoter in both human hepatoma cell lines, Hep3B and HuH-7. Furthermore, we showed that the HBV enhancer sequence not only stimulated the HBcAg gene promoter activity, but also stimulated the HBsAg gene promoter activity in both Hep3B and HuH-7 cells. The enhancer sequence increased the HBsAg gene promoter activity 20-fold in both Hep3B and HuH-7 cell lines, while the increase of the HBcAg gene promoter was 2- and 8-fold in Hep3B and HuH-7 cells, respectively.
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PMID:The enhancer sequence of human hepatitis B virus can enhance the activity of its surface gene promoter. 303 92

We have isolated and characterized a 2.5-kilobase pairs genomic DNA fragment which includes the 5'-flanking region and the first and second exons of the human apolipoprotein (apo) A-I gene. The major transcriptional start site was determined by primer extension analysis and is 235 base pairs (bp) upstream from the AUG translational start codon in liver and 234 bp upstream in the intestine. TATA box-like and CAT box-like sequences and two GC box sequences are present in the intestine 30, 108, 220, and 440 bp upstream, respectively, from the transcriptional start site. Fragments of 570 bp (-487 to +71) and 2.15 kilobase pairs (-2067 to +99) containing the 5'-flanking region of the apoA-I gene were fused upstream to the bacterial chloramphenicol acetyltransferase (CAT) gene. These constructs, designated pA-I(0.6)CAT and pA-I(2.2)CAT, respectively, were introduced into human oral epithelial cells (KB), mouse NIH 3T3 cells, Chinese hamster ovary (CHO) cells, human hepatoma cells (Hep G2), human duodenal epithelial cells (Hutu 80), and human colonic epithelial cells (Caco-2) by calcium phosphate coprecipitation. When compared with control vectors, highly efficient CAT expression of both the pA-I(0.6)CAT and pA-I(2.2)CAT constructs were observed only in cells derived from the liver (Hep G2) and intestine (Caco-2), which is consistent with the tissue specificity of expression of the native gene. Analysis of deletion mutants of the human apoA-I 5'-flanking region revealed that: 1) the region from -250 to -199 bp, from -487 to -413 bp, and -1021 to -691 bp upstream from the transcriptional start site contain sequences required for maximum gene expression; and 2) the regions from -2067 to -1476 bp and -199 to -80 bp contain the sequences required for tissue-specific repression of apoA-I gene expression in non-apoA-I producing cells.
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PMID:Tissue-specific expression of apolipoprotein A-I (ApoA-I) is regulated by the 5'-flanking region of the human ApoA-I gene. 314 80

The human apolipoprotein B (apoB) gene codes for two related proteins, apoB-100 and apoB-48. ApoB-100 is synthesized in the liver, is the major protein constituent of low density lipoprotein, and serves as the ligand for the LDL receptor. cis-acting DNA sequence elements required for hepatic specific apoB transcription were identified in hepatoma (HepG2) and epithelial carcinoma (HeLa) cell lines transfected with apoB/CAT (chloramphenicol acetyltransferase) hybrid constructions. HepG2 cells express the transfected apoB constructions at high levels relative to expression in HeLa cells. Mutational analysis of the 5'-flanking region of the apoB gene revealed the presence of positive and negative regulatory regions. The most distal of these regions, located from -261 to -128 (with respect to the start site of transcription), was found to have a roughly equivalent negative activity in both cell types. However, sequences located from -128 to -86 showed a positive activity in HepG2 cells and a negative activity in HeLa cells. Finally, a sequence element located between positions -86 and -70 was found to have a very strong positive effect in HepG2 cells and only a mild positive effect in HeLa cells. These two proximal regions located between -128 and -70 appear to act together to determine the cell type-specific expression of the apoB gene in HepG2 and HeLa cells. Using the gel mobility shift assay and the DNase I footprinting technique, we demonstrated that DNA binding proteins from HepG2 and mouse liver nuclear extracts interact with the crucial positive region located between -86 and -70. This region was also found to contain sequence elements similar to sequences found in the promoters of other apolipoprotein genes, as well as other genes that are expressed in the liver, suggesting that these genes may share some transcriptional regulatory components.
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PMID:Cell type-specific expression of the human apoB gene is controlled by two cis-acting regulatory regions. 316 76

The trout metallothionein (MT) genes consist of two members. We describe the structure of the first fish MT (tMT-B) gene which shows an overall resemblance but some remarkable differences with mammalian MT genes. The similarities included (i) tripartite structure of the gene, (ii) conservation of cysteine residues, and (iii) a TATAAA signal and two copies of metal-responsive elements (MREs). The differences consisted of (i) an AT-rich tMT-B promoter compared with highly GC-rich mammalian MT promoters and (ii) a lack of SP1-binding sites in the tMT-B promoter. Functional analysis of the tMT-B 5'-flanking region following fusion with the bacterial chloramphenicol acetyltransferase gene and its transfection into the rainbow trout hepatoma cell line revealed that sequences from positions -600 to +8 are sufficient for regulation by metals. Further deletion analyses of this fragment suggested that a minimum of 100 nucleotides upstream of the transcription initiation site are required for induction by cadmium and zinc. The tMT-B promoter was also functional in the human hepatoblastoma cell line, suggesting that an MT regulatory factor(s) is conserved in phylogenetically distant species like humans and fish.
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PMID:Structure of the rainbow trout metallothionein B gene and characterization of its metal-responsive region. 318 57

We have analyzed the cis-acting regulatory DNA elements of the placental rat glutathione S-alkyltransferase (GST-P) gene. Various regions of the 5' flanking sequence were fused with a bacterial chloramphenicol acetyltransferase gene. The transcriptional activity of each construct was determined by the transient expression assay after introduction into a hepatoma cell line. Multiple regulatory elements were identified. Two enhancing elements were located 2.5 and 2.2 kilobases upstream from the transcription start site and designated GST-P enhancers I and II (GPEI and GPEII, respectively). A consensus sequence of the phorbol 12-O-tetradecanoate 13-acetate responsive elements was present in the GPEI and at position -61. GPEII contained two of the simian virus 40 and one of the polyoma enhancer core-like sequences. A silencing element was also found 400 base pairs upstream from the cap site. In accordance with the above observation, endogenous GST-P gene was found to be stimulated when the rat fibroblast line 3Y1 was treated with phorbol 12-O-tetradecanoate 13-acetate. Phorbol 12-O-tetradecanoate 13-acetate enhanced the expression of the transfected GST-P gene to a much higher degree in HeLa cells than in the hepatoma cells, which constitutively expressed the endogenous GST-P. The results are discussed in terms of the specific derepression of GST-P gene during hepatocarcinogenesis in the rat.
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PMID:Multiple regulatory elements and phorbol 12-O-tetradecanoate 13-acetate responsiveness of the rat placental glutathione transferase gene. 320 Aug 31

The tissue-specific expression of the liver-specific rat albumin gene promoter was examined after transfer to various hepatic and non-hepatic cell lines. A 402 base pair sequence from the albumin gene 5' flank enabled a fused reporter chloramphenicol acetyltransferase gene to be expressed in rat hepatoma cell lines but not in fibroblast lines or dedifferentiated hepatoma cells. However, when this same construct was analyzed in permanently transfected cell populations, it was expressed equally well in differentiated and dedifferentiated hepatoma cells and in two of three fibroblast lines tested. The inappropriate expression of the albumin promoter was also seen using the HSV tk gene and the E. coli gpt gene as reporters, and when assayed by colony formation in HAT medium (tk gene) or by S1 protection of transcripts in cotransfected populations (tk and gpt genes). These results show that gene regulatory elements can behave differently in transient vs. stable transfections, and suggest that chromosomal integration can provide long range positive influences on gene expression.
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PMID:The rat albumin gene promoter is appropriately regulated in transient but not in stable transfections. 321 43


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