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)

Aromatase, a cytochrome P-450, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. DNA sequence analysis of the human aromatase gene has revealed that a putative promoter sequence exists immediately up-stream of the second exon. Chloramphenicol acetyltransferase functional analyses of cells transfected with chloramphenicol acetyltransferase expression plasmids containing various DNA fragments derived from the 3'-end of the first intron of the aromatase gene were performed to show that a promoter indeed exists in this region. However, in all of the cell lines used in this study, MCF-7, JAR, OVCAR-3, and skin fibroblast, the function of this promoter was inhibited by a negative regulatory element situated up-stream from the promoter. The results further suggest that this inhibitory element behaves as a silencer element, in that it could inhibit a simian virus-40 promoter from a distance of several kilobases. This negative element worked in both orientations and inhibited the functions of several promoters, including the newly identified promoter situated in the 3'-end of the first intron of the human aromatase gene. Primer extension analysis has been performed to determine the potential transcription start site. The mechanism of the regulation of aromatase expression is known to be very complex. The presence of a promoter and a silencer at the 3'-end of the first intron may represent one additional way that aromatase expression is controlled in estrogen-producing cells.
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PMID:Identification of a promoter and a silencer at the 3'-end of the first intron of the human aromatase gene. 133 79

The regulation of the core promoter of Hepatitis B virus (HBV) was investigated using the chloramphenicol acetyltransferase (CAT) reporter system. Deletional analysis of sequences 5' to the HBV core promoter indicated the presence of a negative regulatory element (NRE) located within a 282-bp BamHI-HincII DNA fragment. The NRE was functional in hepatic as well as nonhepatic cells. Results of in vivo competition experiments suggest a role for cellular transacting repressor protein(s) in the functioning of the NRE. The HBV NRE, positioned 5' to the SV40 early promoter, inhibited the activity of the heterologous promoter in an orientation-independent, but position-dependent manner. These data indicate that the HBV NRE is a silencer element, which functions to downregulate the activity of the core promoter.
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PMID:Hepatitis B virus C gene promoter is under negative regulation. 160 26

Simian virus (SV) 40 enhancer (nucleotide position 108 to 294) was combined with chloramphenicol acetyltransferase (CAT) plasmid whose expression is under control of mouse DNA polymerase beta gene promoter. Although the SV40 enhancer stimulated the transient CAT-expression directed by the DNA polymerase beta gene promoter two to three fold in human HeLa cells, it repressed the CAT-expression by 50 to 60% in mouse NIH/3T3 cells. The repression was observed relatively independently on the orientation of the insertion and the distance from the promoter. These properties of the enhancer are very similar to those of so-called transcriptional silencer element. In both HeLa and NIH/3T3 cells, the SV40 enhancer stimulated effectively its own early gene promoter-directed CAT-expression. In mouse immature T-cell line RV-1 in which the SV40 promoter-enhancer did not function, no effect of the SV40 enhancer sequence on the DNA polymerase beta promoter-directed CAT-expression was observed. Thus, it is suggested that both cell type-specific trans-acting factor(s) and the specific combination with the promoter sequence turn the properties of the SV40 enhancer into those of a silencer.
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PMID:Paradoxical effect of Simian virus 40 enhancer on the function of mouse DNA polymerase beta gene promoter. 254 52

We have studied the 5'-flanking sequences required for the transcriptional regulation of human epsilon-globin gene expression. A series of deletion mutants of the human epsilon-globin gene 5'-flanking sequences were constructed and linked to the bacterial chloramphenicol acetyltransferase gene. Expression of these constructs was tested in HeLa cells and the human erythroleukemia K-562 cells. By measuring chloramphenicol acetyltransferase activities and mRNA levels we found that the sequence between -177 and -392 base pairs (bp) relative to the mRNA initiation site exerts a negative effect on epsilon-globin promoter activity. This effect is more pronounced in HeLa cells compared with K-562 cells. To further characterize the negative control region we cloned the DNA sequence between -177 and -392 bp either 5' or 3' of the epsilon-globin promoter and in either orientation. Our data indicate that this negative control region inhibits the epsilon-globin promoter activity in a position- and orientation-independent manner, thus suggesting that it is a silencer. In addition, the silencer also inhibits the expression from the Herpesvirus thymidine kinase promoter. Sequence comparison reveals that there are three short regions within the silencer that share extensive homology with those found in other negative control DNA elements. Our results therefore indicate that an upstream silencer element is present in the epsilon-globin gene and that it may play an important role in the control of epsilon-globin gene expression during development.
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PMID:Identification of a transcriptional silencer in the 5'-flanking region of the human epsilon-globin gene. 274 86

A portion of the 5'-flanking region of the glycoprotein IIb (alpha IIb) integrin gene extending from -598 to +32 base pairs was isolated. This DNA segment is capable of driving low level base-line transcription in undifferentiated K562 cells. It also contains elements which direct the markedly increased expression observed following megakaryocytic differentiation of K562 cells with phorbol dibutyrate. Analysis of hybrid alpha IIb-chloramphenicol acetyltransferase reporter gene constructs indicates that at least three regions within the -598 to +32 region control differentiation-dependent alpha IIb transcription. Two enhancer elements as well as a silencer domain all regulate chloramphenicol acetyltransferase transcriptional activity in K562 cells. Gel mobility shift experiments revealed that nuclear binding proteins are able to interact with all three DNA regions. A small region lying between -124 and -99 bases is able to bind to nuclear proteins in undifferentiated cells but not in differentiated cells as evidenced by gel mobility shift and foot-printing studies and corresponds to the silencer element identified in the functional studies. Therefore, the tissue-specific expression of alpha IIb may be controlled transcriptionally by both positive and negative factors with the silencer element playing a major role in regulating differentiation-dependent expression.
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PMID:Transcriptional regulation of alpha IIb integrin gene expression during megakaryocytic differentiation of K562 cells. Role of a silencer element. 751 32

The cyclic AMP (cAMP)-inducible promoter from the rat lactate dehydrogenase A subunit gene (LDH A) is associated with a distal negative regulatory element (LDH-NRE) that represses inherent basal and cAMP-inducible promoter activity. The element is of dyad symmetry, consisting of a palindromic sequence with two half-sites, 5'-TCTTG-3'. It represses the expression of an LDH A/chloramphenicol acetyltransferase (CAT) reporter gene in a dose-dependent, orientation- and position-independent fashion, suggesting that it is a true silencer element. Uniquely, it selectively represses cAMP-responsive element (CRE)-dependent transcription but has no effect on promoters lacking a CRE sequence. The repressing action of LDH-NRE could be overcome by cotransfection with LDH A/CAT vector oligonucleotides containing either the LDH-NRE or CRE sequence. This suggests that the reversal of repression was caused by the removal of functional active, limiting transacting factors which associate with LDH-NRE as well as with CRE. Gel mobility shift, footprinting, and Southwestern blotting assays demonstrated the presence of a 69-kDa protein with specific binding activity for LDH-NRE. Additionally, gel supershift assays with anti-CREB and anti-Fos antibodies indicate the presence of CREB and Fos or antigenically closely related proteins with the LDH-NRE/protein complex. We suggest that the LDH-NRE and CRE modules functionally interact to achieve negative modulation of cAMP-responsive LDH A transcriptional activity.
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PMID:Identification of a silencer module which selectively represses cyclic AMP-responsive element-dependent gene expression. 756 66

The biosynthesis of estrogens is catalyzed by aromatase P450 (P450arom), the product of the CYP19 gene. The tissue-specific expression of the CYP19 gene is regulated by means of tissue-specific promoters through the use of alternative splicing mechanisms. Thus, transcripts containing various 5'-untranslated termini are present in human placenta and other fetal tissues, ovary, brain, and adipose stromal cells. Sequence corresponding to untranslated exon 1.4 is present in 5'-termini of transcripts expressed in adipose tissue and fetal liver, as well as adipose stromal cells in primary culture in the presence of dexamethasone and fetal calf serum (FCS). Identification of hormone-responsive, tissue-specific promoter regions, as well as growth factor-response elements upstream of exon 1.4, may provide insight into the regulation of estrogen biosynthesis in adipose tissue, which is implicated in the development of breast and endometrial cancer. The goals of the present study were to define the 1.4 promoter region with respect to the start of transcription and to characterize the region(s) responsible for conferring glucocorticoid responsiveness on aromatase expression. The transcription initiation site was identified by means of primer extension and S1 nuclease protection analyses. No TATA-like sequence was evident upstream of this site. Various deletion mutations of the upstream flanking region of exon 1.4 and including part of exon 1.4 were made using polymerase chain reaction or restriction enzyme digestion. The genomic fragments were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These constructs were transfected into adipose stromal cells and fetal hepatocytes in primary culture in medium containing FCS with or without dexamethasone. The -560/+10 base pair (bp) construct expressed CAT activity after a putative silencer element was deleted, and expression was induced by dexamethasone about 3-fold. Transfection of the -330/+170 bp construct, which contains an upstream glucocorticoid response element (GRE) as well as an Sp1-like sequence in untranslated exon 1.4, resulted in an 8-fold stimulation of expression of CAT activity by dexamethasone. The upstream GRE as well as the Sp1-like sequence in untranslated exon 1.4 were mutated separately, and together, to further confirm whether the GRE or Sp1 binding site play a role in the regulation of promoter 1.4-driven transcription. Mutation of either the GRE or Sp1 binding site, or both, in the -330/+170 bp construct, resulted in loss of dexamethasone-induced CAT reporter gene expression.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the sequences of the human CYP19 (aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes. 777 80

Transfection experiments with constructs containing various 5'-deleted fragments of the human lipoprotein lipase (LPL) promoter and the chloramphenicol acetyltransferase reporter gene revealed an LPL silencer element (LSE) in the region of nucleotides -225 to -81 of the LPL gene that functioned in Chinese hamster ovary (CHO) and HeLa cells. Gel retardation competition analysis showed the presence of a nuclear factor(s) capable of binding to the sequence of nucleotides -169 to -152 of LSE (LSE-6) in a single-stranded (opposite-strand) and double-stranded specific fashion, the binding affinity being almost the same in the two binding forms. Site-directed mutagenesis indicated that almost the entire sequence of LSE-6 was necessary to form the complexes and also critical for silencing activity in CHO cells. The amounts of this binding factor(s) in CHO and HeLa cells were closely associated with transcriptional silencing activity. Photochemical cross-linking experiments indicated that the single- and double-stranded elements recognized the same binding factor(s) with molecular masses of 54 to 63 kDa and 109 to 124 kDa. The 109- to 124-kDa DNA binding factor(s) was found to be a doublet of that of the 54- to 63-kDa factor by isoelectric focusing or by increasing the time of exposure to UV irradiation. When inserted upstream of another gene such as that of the simian virus 40 enhancer/promoter of pSV2CAT, the sequence of nucleotides -190 to -143 (LSE-1) also suppressed transcription of the reporter gene in CHO cells. These results strongly suggest that the LSE plays a role in regulation of LPL gene expression by suppressing its transcription.
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PMID:A silencer element for the lipoprotein lipase gene promoter and cognate double- and single-stranded DNA-binding proteins. 779 60

The Na,K-ATPase is an integral plasma membrane protein consisting of alpha and beta subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional alpha isoform genes, the one encoding the alpha 3 isoform is the most tissue-restricted in its expression, being found primarily in the brain. To identify regions of the alpha 3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5'-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The alpha 3-CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5'-flanking sequence of the alpha 3-CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the alpha 3 isoform gene are present within 210 bp upstream of the transcription initiation site. alpha 3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively acting cis element.
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PMID:The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice. 798 27

Thrombomodulin, a glycoprotein expressed in endothelial cells, has an important role in the blood coagulation system as a modulator. Functional characterization of the 5'-regulatory region of the human thrombomodulin gene was carried out to identify elements necessary for its expression. We used a series of dissected gene constructs containing the bacterial chloramphenicol acetyltransferase gene in transient transfection assays on human umbilical vein endothelial cells. The region extending from -290 to -33 of the 5' end flanking sequence is required for the full expression of this gene. Within this region, four potential Sp1 sites were found, and the sequences of Sp1 sites were mutated to identify their role in the promoter activity of the gene, showing that the two Sp1 sites at -207 and -141 are important for the full activity of the thrombomodulin promoter. Site-directed mutation analysis identified sequence elements GCAATC at -110 as a functioning CAAT box. Another three regions, -290 to -223, -99 to -68, and -67 to -33 have unidentified positively and negatively acting elements. A silencer element was located in the region spanning from -947 to -772 bases of the 5' end flanking region. These data indicate that the expression of the thrombomodulin gene is regulated by various elements which act positively or negatively.
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PMID:Functional characterization of the 5'-regulatory region of the human thrombomodulin gene. 839 36


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