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)

Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically suppress activities of the promoter and enhancer of the human alpha-fetoprotein (AFP) gene in HuH-7 human hepatoma cells as analyzed by transient transfection assays using the chloramphenicol acetyltransferase gene as a reporter. In contrast to the AFP gene, albumin promoter and enhancer activities were not affected by EGF and TPA. Unexpectedly, however, Northern blot analysis revealed that the albumin mRNA level as well as the AFP mRNA level were reduced by treatment with EGF and TPA. We propose that in HuH-7 cells, the AFP enhancer stimulates the albumin promoter as well as the AFP promoter; and consequently, inhibition of the AFP enhancer by EGF and TPA results in reduction of both AFP and albumin mRNA levels. The significance of the involvement of the AFP enhancer in albumin transcription is discussed in relation to the inverse pattern of expression of the AFP and albumin genes in neonatal growth.
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PMID:A possible mechanism of inverse developmental regulation of alpha-fetoprotein and albumin genes. Studies with epidermal growth factor and phorbol ester. 137 Apr 67

A series of simian virus 40-immortalized hepatocyte cell lines which are heterogeneous with regard to expression of albumin protein and RNA were characterized for their ability to transcribe the albumin gene. Nascent chain extension assay showed that albumin RNA levels in these cells were determined predominantly at the transcription level. The albumin promoter and enhancer sequences were fused to the bacterial chloramphenicol acetyltransferase gene; the ability of the resulting expression constructs to drive chloramphenicol acetyltransferase expression after transfection into these hepatocyte cell lines was measured. The activity of the albumin promoter and enhancer constructs in primary hepatocytes was also measured. The albumin promoter was expressed differentially in these cells; however, no correlation was found between the transcriptional efficiency of the transfected albumin promoter and endogenous albumin transcription. The albumin enhancer was functional in some but not all albumin-positive cells. The minimal albumin enhancer was mapped to a 330-base pair fragment extending from -9.94 kilobases (kb) to -10.27 kb; three elements within this fragment recently shown to be necessary for enhancer function in a murine hepatocyte cell line were also essential for albumin enhancer function in the rat hepatocyte cell line CWSV1. A transcriptional silencer was identified which could suppress the expression of the homologous albumin promoter and the heterologous herpes simplex virus thymidine kinase promoter. Preliminary analysis localized the albumin silencer between -11 and -12 kb. Our results suggest that multiple regulatory sequences may act cooperatively to determine efficient tissue-specific expression of the albumin gene.
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PMID:Functional analyses of albumin expression in a series of hepatocyte cell lines and in primary hepatocytes. 141 9

The CYP2C6 gene becomes maximally transcriptionally activated in livers of postpubertal rats. We examined the role of upstream DNA and liver-specific transcription factors in regulation of this promoter by use of transient transfection of heterologous chloramphenicol acetyltransferase gene constructs and vectors containing cDNAs encoding the liver-enriched transcription factors HNF-1 alpha, C/EBP, and DBP. Only DBP was able to activate the CYP2C6 promoter in HepG2 cells. Transactivation was not observed in one mouse and two human nonhepatic origin cell lines tested. Analysis of various constructs in which CYP2C6 upstream DNA was deleted revealed that DNA between -38 to -103 was involved in DBP-mediated activation. A partially purified preparation of DBP produced a footprint between -43 and -64 bp upstream of the transcription start site. A 32P-labeled double-stranded oligonucleotide, containing sequence information corresponding to -40 to -65, bound to both partially pure DBP and extracts from livers of rats as young as 1 week and as old as 25 weeks of age, as assessed by gel mobility shift analysis. This binding was eliminated by coincubation with excess unlabeled -40/-65 double-stranded oligonucleotide and by an oligonucleotide corresponding to the D site of the rat albumin gene. A gel mobility shift-Western immunoblot analysis revealed that the -40/-65 sequence bound to DBP only in liver nuclear extracts from rats older than 3 weeks; maximal binding was observed by 7 weeks of age, and no binding was detected from 1-week-old rat liver extracts. Interestingly, the DBP-binding regions of both CYP2C6 and albumin bind to C/EBP, but this factor is capable of transactivating only the latter gene. Although the DBP-binding regions in these two genes share no obvious sequence similarities, the CYP2C6 region contains consensus palindromic half sites for DBP-related binding proteins and affinity for recombinant DBP of 17-fold greater than that of the D site of albumin. This difference in affinity is probably responsible for the markedly lower amounts of DBP required for half-maximal activation of the CYP2C6 promoter, as compared with the albumin promoter, in transactivation transfection assays. These data indicate that the CYP2C6 gene may be regulated, at least in part, by DBP, a liver transcription factor produced when rats reach puberty that may also be involved in maintenance of albumin gene transcription.
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PMID:Role of the liver-enriched transcription factor DBP in expression of the cytochrome P450 CYP2C6 gene. 158 73

We studied the effects of transfection of the normal c-Ha-ras gene, rasGly-12, and its oncogenic mutant, rasVal-12, on expression of the alpha-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasVal-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasVal-12 suppressed the activity of enhancer and promoter regions containing A + T-rich sequences (AT motif). In contrast, rasVal-12 did not affect the promoter activity of the albumin and human hepatitis B virus pre-S1 genes even though these promoters contain homologous A + T-rich elements. ras transfection appeared to induce phosphorylation of nuclear proteins that interact with the AFP AT motif, since gel mobility analysis revealed the formation of slow-moving complexes which was reversed by phosphatase treatment. However, similar changes in complex formation were observed with the albumin and hepatitis B surface antigen pre-S1 promoters. Therefore, this effect alone cannot explain the specific down regulation of the AFP promoter and enhancer activity. ras-mediated suppression of the AFP gene may reflect the process of developmental gene regulation in which AFP gene transcription is controlled by a G-protein-linked signal transduction cascade triggered by external growth stimuli.
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PMID:c-Ha-ras down regulates the alpha-fetoprotein gene but not the albumin gene in human hepatoma cells. 169 Aug 41

Expression of genes encoding transferrin and the vitamin D-binding protein is described in a cell line, U-2 OS, derived from a human osteogenic sarcoma. The mRNA transcripts of transferrin and vitamin D-binding protein were shown to be the lengths of those found in normal human liver. The cells synthesize and secrete the transferrin and vitamin D-binding proteins, in addition to human albumin and ceruloplasmin. The U-2 OS cells were successfully transfected with chimeric genes carrying 670 bp of the 5' regulatory sequence of the human transferrin gene fused to a reporter chloramphenicol acetyltransferase gene. These data indicate that the appropriate transcriptional factors required for expression of four plasma proteins are produced by U-2 OS nuclei and that the U-2 OS cell line will be useful for studies analyzing regulation of these genes.
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PMID:Expression of transferrin and vitamin D-binding protein genes in an osteogenic sarcoma cell line. 229 48

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.
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PMID:Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes. 245 90

DNase I footprinting and gel mobility shift analysis showed that an HuH-7 hepatoma nuclear protein, termed AFP1, binds specifically to an AT-rich sequence, TGATTAATAATTACA, in domain B of the human alpha-fetoprotein enhancer. No such binding activity was found in HeLa cell nuclei. Transient transfection studies showed that a 54-base-pair region corresponding to the AFP1-binding site could stimulate the simian virus 40 early promoter to express a linked chloramphenicol acetyltransferase gene in an orientation-independent and cell-specific manner. The correlation between the binding of AFP1 and the stimulation of chloramphenicol acetyltransferase gene expression strongly suggests that specific interaction of AFP1 with the AT motif is important for cell-specific transcriptional enhancement. Competition gel mobility shift analysis revealed that similar AT-rich sequences with high affinities to AFP1 were also present in the promoters of the alpha-fetoprotein and albumin genes. These results suggest that AFP1 may function as a common regulatory factor in the transcription of the alpha-fetoprotein and albumin genes.
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PMID:Interaction of a hepatoma-specific nuclear factor with transcription-regulatory sequences of the human alpha-fetoprotein and albumin genes. 246 95

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

We present evidence that a foreign gene driven by natural mammalian regulatory elements can be targeted to hepatocytes and the resultant gene expression made to persist. This was accomplished using a soluble DNA carrier system consisting of two covalently linked components: 1) a polycation, poly-L-lysine, that can bind DNA in a strong but non-damaging interaction, and 2) an asialoglycoprotein which can be targeted specifically to hepatocytes by cell surface asialoglycoprotein receptors unique to this cell type. A plasmid, palb-CAT, containing the gene for chloramphenicol acetyltransferase (CAT) driven by mouse albumin regulatory sequences was complexed to the carrier system. Intravenous injection of palb-CAT DNA in the form of a complex resulted in the presence of CAT enzyme activity in liver homogenates 24 h after injection. The targeted gene expression, however, was transient, reaching a maximum of 10 units/g liver at 24 h but was not detectable by 96 h. However, partial hepatectomy 30 min after injection resulted in persistent high levels of hepatic CAT activity (11.3 units/g) through 11 weeks post-injection. Southern analysis of livers 11 weeks after partial hepatectomy demonstrated that some of the targeted DNA had been integrated into the host genome. We conclude that a foreign gene driven by natural mammalian regulatory elements can be delivered to hepatocytes by intravenous injection in vivo using a soluble DNA carrier system. Foreign gene expression targeted in this manner can be made to persist by stimulation of hepatocyte replication.
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PMID:Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. 279 40

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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