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)

We have characterized a cDNA and the corresponding gene for a cyclic AMP-inducible gene expressed during Dictyostelium development. This gene, BP74, was found to be first expressed about the time of aggregate formation, approximately 6 h after starvation. Accumulation of BP74 mRNA did not occur in Dictyostelium cells that had been starved in fast-shaken suspension cultures but was induced in similar cultures to which cyclic AMP pulses had been added. The BP74 cDNA and gene were characterized by DNA sequence analysis and transcriptional mapping. When the BP74 promoter region was fused with a chloramphenicol acetyltransferase reporter gene and reintroduced into Dictyostelium cells, the transfected chloramphenicol acetyltransferase gene displayed the same developmentally regulated pattern of expression as did the endogenous BP74 gene, suggesting that all of the cis-acting elements required for regulated expression were carried by a 2-kilobase cloned genomic fragment. On the basis of sequence analysis, the gene appeared to encode a protein containing a 20-residue hydrophobic sequence at the amino-terminal end and 26 copies of a 20-amino-acid repeat.
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PMID:Expression and organization of BP74, a cyclic AMP-regulated gene expressed during Dictyostelium discoideum development. 255 85

Tyrosine hydroxylase (TH) is selectively expressed in catecholaminergic neurons and in chromaffin cells of the adrenal medulla. Constructs in which 5' flanking sequences of the rat TH gene directed expression of bacterial chloramphenicol acetyltransferase (CAT) were transfected into cell lines and assayed for transient expression of CAT. In most nonexpressing cell lines, CAT levels were less than 5% of that found in a TH-positive pheochromocytoma line (PC8b). In two lines described here, a rat anterior pituitary cell line (GH4) and a rat fibroblast line (Fr3T3), CAT expression reached 12 and 20%, respectively, of the PC8b level. Greater than 90% of the PC8b activity was lost when sequences between -212 and -187 (in relation to the transcriptional initiation site) were deleted. Further deletions that removed the cyclic AMP response element (CRE) (-45) and the TATA box at -29 reduced transcriptional activity to background in all three lines. These data suggest that 212 nucleotides of the 5' sequence are sufficient for pheochromocytoma expression and that information between -212 and -187, which includes an AP1 site (-206 to -200), is essential for full transcriptional activity. In addition, sites for other protein transcription factors (AP2, POU/Oct, SP1, and CRE) reside between -221 and -38 and are largely conserved between the human and rat gene.
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PMID:5' flanking DNA sequences direct cell-specific expression of rat tyrosine hydroxylase. 257 79

We are interested in the mechanisms by which endocrine and developmental factors regulate TSH synthesis at both pre-translational and post-translational levels. Thyroid hormone profoundly decreases transcription of the TSH-beta gene, while TRH and agents modifying cyclic AMP increase transcription. To elucidate the molecular mechanisms underlying these effects, human embryonal kidney cells were transfected with constructs of the human TSH-beta gene fused to the chloramphenicol acetyltransferase gene. The first exon of human TSH-beta, contains an element that increases basal expression and mediates T3-induced gene repression, probably through a direct interaction with c-erbA beta. This transcriptional repression by T3 appears aberrant in thyrotropic tumors. In contrast, TRH and agents modifying cyclic AMP mediate increased transcription of TSH-beta through interacting with upstream regulatory elements. Thyroid hormone, TRH and developmental factors also regulate the branching pattern and relative sialylation of TSH carbohydrate chains, which may affect TSH action in vitro and in vivo. Certain thyrotropic tumors produce TSH with more complex carbohydrate branching patterns, which may increase its biologic activity.
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PMID:Pre-translational and post-translational regulation of TSH synthesis in normal and neoplastic thyrotrophs. 269 12

We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the murine Sparc (osteonectin) gene in parietal endoderm cells and in F9 embryonal carcinoma cells induced to differentiate into parietal endoderm with retinoic acid and cyclic AMP. Varying lengths of flanking sequences extending up to 3.0 kilobase pairs 5' of the transcription initiation site were linked to the bacterial chloramphenicol transacetylase gene in the Bluescript M13- vector. The constructs were tested in transient assays, using a beta-galactosidase plasmid as a transfection control. Sequences between 78 and 169 base pairs upstream of the cap site are the minimum required for cell-type specific promoter activity; this region is dominated by two oligopurine/oligopyrimidine stretches or "GAGA" boxes and is highly conserved between the mouse and bovine genes. Addition of the sequence between -169 and -449, which includes part or all of a third GAGA box, results in increased parietal endoderm specific transcription, up to a maximum of 6.3-fold higher than in undifferentiated F9 cells. Further addition of sequences between -449 and -638 markedly reduces promoter activity in both cell types but parietal endoderm-specific activity is restored in constructs containing 2.2 and 3.0 kilobase pairs of flanking DNA. In addition, we have identified sequences related to the consensus sequence for steroid response elements, one of which is able to confer progesterone-enhanced transcription when tested with a heterologous promoter in steroid responsive cells. These results suggest that negative and positive elements normally interact to regulate the temporal and tissue-specific patterns of Sparc gene transcription seen in vivo.
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PMID:Evidence for positive and negative regulatory elements in the 5'-flanking sequence of the mouse sparc (osteonectin) gene. 274 36

From a mouse genomic DNA library we have isolated sequences containing the entire coding region for histone H1(0) mRNA, flanked by several kb at both the 3' and 5' ends. Deletions of the 5' upstream region ligated to the chloramphenicol acetyltransferase (CAT)-encoding gene as a reporter, have shown that a region from bp -400 to -600 is necessary and sufficient for efficient transcription. We have also shown that treatment of F9 teratocarcinoma cells with retinoic acid and cyclic AMP (which differentiates F9 cells to parietal endoderm) clearly increases CAT activity several times over the level found in untreated F9 cells. This increase was observed in transient, as well as in stably transfected cells. Analysis of the deletions in differentiating cells indicates that the element responsible for the observed increase in CAT activity, is contained within the first 700 bp upstream from the H1(0) mRNA cap site.
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PMID:Cloning and characterization of the mouse histone H1(0) promoter region. 280 18

The promoter region of mouse lactate dehydrogenase-A gene was fused with the chloramphenicol acetyltransferase gene of Escherichia coli, and the expression of this fusion gene was induced by cyclic AMP in Chinese hamster ovary wild-type cells, but not in mutants affecting the regulatory or catalytic subunit of cAMP-dependent protein kinase.
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PMID:Cyclic AMP-induced expression of the mouse lactate dehydrogenase-A promoter-cat fusion gene in Chinese hamster ovary wild-type cells, but not in cAMP-dependent protein kinase mutant cells. 282 Apr 3

The alpha subunit of the placental hormone chorionic gonadotropin is regulated by cyclic AMP (cAMP) at the transcriptional level. A cAMP-responsive fusion gene (alpha-CAT) containing 1.5 kilobases of the alpha gene 5'-flanking sequence linked to the chloramphenicol acetyltransferase (CAT) gene was used as a transcriptional reporter in competition assays in transfected JEG-3 choriocarcinoma cells. Expression of the alpha-CAT fusion gene increased linearly with increasing amounts of transfected plasmid and was maximal at the same amount of alpha-CAT DNA (2 micrograms) with or without cAMP treatment. Various amounts of different competitor DNA sequences were cotransfected with the alpha-CAT reporter plasmid to examine the interactions of intracellular trans-acting factors with the regulatory elements of the alpha gene promoter. An 800-base-pair fragment of alpha gene 5'-flanking sequence inhibited both basal and cAMP-stimulated transcription of the alpha-CAT reporter plasmid in a dose-dependent manner, indicative of interactions with one or more trans-acting factors that activate alpha gene expression. The alpha gene sequences that interact with intracellular regulatory factors were defined by using several discrete regions of the 5'-flanking sequence as competitors for alpha-CAT expression. A proximal promoter sequence (-99 to +44) containing the CCAAT box, TATA box, and transcriptional initiation site was a relatively ineffective competitor of alpha-CAT transcription. In contrast, an upstream sequence between -236 and -100 was an effective competitor for transcriptional activators of alpha-CAT expression. Competition for alpha-CAT expression by this regulatory sequence did not require cis interactions with downstream promoter elements and was equally effective with or without cAMP treatment. An 18-base-pair repeated sequence within this region of the alpha gene (-146 to -111) greatly enhanced both basal gene expression and cAMP responsivity and also competed for limiting cellular transcription factors. These findings suggest that JEG-3 cells contain trans-acting factors that interact with a cAMP response element to activate alpha gene transcription. The chorionic gonadotropin beta gene 5'-flanking sequence also competed for alpha-CAT expression, suggesting that a common trans-acting factor is shared by the regulatory sequences of the alpha and beta genes.
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PMID:trans-acting factors interact with a cyclic AMP response element to modulate expression of the human gonadotropin alpha gene. 282 16

The chronic effect of cAMP-dependent regulation on adrenocortical steroidogenesis is known to be revealed in the stimulation of the biosynthesis of steroidogenic enzymes. P-450(SCC), one of the enzymes, catalyzes the first and the rate-limiting reaction in steroidogenesis from cholesterol and its synthesis is regulated by cAMP. In order to investigate cis-acting DNA elements of this gene in response to cAMP-dependent regulation, we have constructed a fusion gene (pSCC5.4k) by ligating the 5'-flanking and the upstream untranslated region (5.4 kb) of the human P-450(SCC) gene to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected it into various culture cells including Y-1 (mouse adrenal tumor), L929 (mouse fibroblast), HTC (rat hepatoma) and Hepa-1 (mouse hepatoma). Only Y-1 cells transfected with pSCC5.4k were found to express transiently the enhanced CAT activity in response to the cAMP analogue, cyclic dibutyryl-AMP (Bt2cAMP). Primer-extension analysis of RNA prepared from the cells treated with or without Bt2cAMP showed that the enhanced CAT activity was due to an increase in the CAT mRNA and that the transcription start site, determined here with the human P-450 gene in the adrenal cortex, was correctly utilized with the fusion gene in the transient expression system. Forskolin and cholera toxin, activators of adenylate cyclase, also increased the expression of the CAT activity in the Y-1 cells. It has been demonstrated, therefore, that the cAMP-dependent regulation of the P-450(SCC) gene in adrenal cortex is faithfully reflected in the transient expression system using Y-1 cells and the fusion gene and that a cis-acting DNA element(s) in response to cAMP is present within the 5'-flanking sequence (5.4 kb) of the P-450(SCC) gene.
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PMID:The 5'-flanking region of the human P-450(SCC) gene shows responsiveness to cAMP-dependent regulation in a transient gene-expression system of Y-1 adrenal tumor cells. 283 Oct 49

Fusion genes containing segments of the promoter region of the human LDL receptor gene and the coding sequence of the bacterial enzyme, chloramphenicol acetyltransferase (CAT), were introduced into JEG-3 human choriocarcinoma cells. Constructs containing 177 base pairs of 5'-flanking DNA (pLDLR-CAT 234) or 6500 base pairs (pLDLR-CAT 6500) promoted CAT activity in transient expression assays. Although both pLDLR-CAT 234 and pLDLR-CAT 6500 contain sequences related to the recently reported consensus sequence for cyclic AMP responsiveness, treatment of the transfected JEG-3 cells with 8-bromo-cAMP did not increase CAT activity. The cyclic AMP analog did, however, stimulate steroidogenesis and hCG secretion and increase CAT activity in cells transfected with p18X2SV1CAT, which contains two copies of an 18 base pair sequence corresponding to the cAMP-responsive element of the human alpha chorionic gonadotropin gene.
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PMID:The upstream promoter of the human LDL receptor gene does not contain a cyclic AMP response element. 283 85

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.
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PMID:Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. 285 Apr 95


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