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 investigated the ability of a constitutively active Gq-alpha mutant, Q209L-alpha q, to regulate target gene expression. Transient expression in GH3 pituitary cells of a rat proximal prolactin promoter-chloramphenicol acetyltransferase construct (-187)PRL-CAT, was stimulated by co-expression of Q209L alpha q, but not by wild-type alpha q. Q209L-alpha q stimulated expression of constructs driven by promoters for either rat prolactin or growth hormone, but not of a control construct driven by the thymidine kinase promoter. Thus, transcriptional effects of alpha q are specific both for the activated state of this G-alpha subunit and the promoter examined. Since both the prolactin and growth hormone promoters are activated by the pituitary cell-specific transcription factor Pit-1, we examined whether a Pit-1 binding site could direct a response to Q209L-alpha q. Two copies of prolactin promoter Pit-1 binding site 1P conferred upon a heterologous metallothionein promoter a response to Q209L-alpha q, implying an involvement of this site in the transcriptional action of Q209L-alpha q on the prolactin promoter. The phorbol ester activator of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate, stimulated (-187)PRL-CAT activity, but opposed the action of Q209L-alpha q on activity of this PRL-CAT construct. Q209L-alpha q stimulation of (-187)PRL-CAT activity was inhibited by co-expression of a dominant negative Raf mutant, Raf-C4, but not by a point mutant of Raf-C4 with reduced inhibitory properties. These results imply that activated alpha q subunits can stimulate prolactin promoter activity via a pathway that involves a Pit-1 DNA binding site(s), is opposed by protein kinase C, and is mediated by a pathway in which Raf-1 kinase plays a role.
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PMID:Constitutively active Gq-alpha stimulates prolactin promoter activity via a pathway involving Raf activity. 748 29

We studied reporter gene expression in synovial tissue after intra-articular administration of an expression plasmid into the knees of rabbits and rats. In both species, administration of a plasmid encoding beta-galactosidase led to gene expression in the synovial cells lining the joint. Expression correlated with the presence of plasmid DNA in synovial tissue extracts. Studies with a plasmid encoding chloramphenicol acetyltransferase demonstrated that gene expression persists for 2-5 days after administration. Southern blotting demonstrated that the administered plasmid was taken up rapidly by synovial tissue and degraded. By 24 hr after administration, no intact plasmid could be detected by Southern blotting, although small amounts of plasmid could be amplified by PCR up to 7 days. Administration of a plasmid encoding human growth hormone demonstrated that this product could be expressed from synovial cells and secreted into the synovial fluid. The histological distribution of gene expression in synovium resembles the known distribution of particulate materials injected into the joint and suggests that plasmid DNA is taken up by nonspecific endocytosis like other particulate materials during the remodeling of synovial fluid.
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PMID:Gene transfer to synovial cells by intra-articular administration of plasmid DNA. 757 97

A major metabolite of the vitamin D analogue 1 alpha-hydroxyvitamin D2 in human liver cells in culture has been identified as 1 alpha,24(S)-dihydroxyvitamin D2 [1 alpha,24(S)-(OH)2D2]. 1 alpha-Hydroxyvitamin D3 incubated with the same cells gives rise to predominantly 25- and 27-hydroxylated products. Our identification of 1 alpha,24(S)-dihydroxyvitamin D2 is based on comparisons of the liver cell metabolite with chemically synthesized 1 alpha,24(S)-(OH)2D2 and 1 alpha,24(R)-(OH)2D2 by using HPLC, GC and GC-MS techniques. The stereochemical orientation of the 24-hydroxyl group was inferred after X-ray-crystallographic analysis of the 24(R)-OH epimer. 1 alpha,24(S)-Dihydroxyvitamin D2 binds strongly to the vitamin D receptor and is biologically active in growth hormone and chloramphenicol acetyltransferase reporter gene expression systems in vitro, but binds poorly to rat vitamin D-binding globulin, DBP. We suggest that this metabolite, 1 alpha,24(S)-(OH)2D2, possesses the spectrum of biological properties to be useful as a drug in the treatment of psoriasis, metabolic bone disease and cancer.
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PMID:1 alpha,24(S)-dihydroxyvitamin D2: a biologically active product of 1 alpha-hydroxyvitamin D2 made in the human hepatoma, Hep3B. 764 51

The transcription rates of the rat serine protease inhibitor 2.3 and 2.1 genes (spi 2.3 and spi 2.1), which are normally very low and high, respectively, are inversely modulated during inflammation. Two growth-hormone-response elements (GHRE-I and GHRE-II) maintain the spi 2.1 gene under the stringent control of growth hormone [Le Cam, A., Pantescu, V., Paquereau, L., Legraverend, C., Fauconnier, G. & Asins, G. (1994) J. Biol. Chem. 269, 21532-21539], whereas spi 2.3 appears to escape control by this hormone, despite the presence in its promoter of a functional GHRE-I. A major difference between these two otherwise very similar genes is the presence in spi 2.3 of a specific 348-bp extension of the 3' untranslated region (3' UTR). Inserting this 3' UTR element downstream of the polyadenylation signal or upstream of the spi 2.3 promoter in constructs containing the chloramphenicol acetyltransferase gene strongly decreases basal transcription and inhibits growth-hormone-stimulated transcription, but poorly affects transcriptional stimulation by dexamethasone or interleukin-6. The spi 2.3 3' UTR extension also inhibits, basal and growth-hormone-induced transcription from the spi 2.1 promoter. Repressor activity appears to be distributed throughout the specific extension of the 3' UTR and seems to involve interactions with two types of 5' cis-acting promoter elements. The first is the GAGA box, a key control spi promoter element, whose mutation faithfully reproduces the effects of the 3' UTR silencer on spi 2.1 and spi 2.3 promoters. The second is represented by CCAAT enhancer-binding-protein-(C/EBP)-binding sites, whose functions are severely impaired by the spi 2.3-specific 3' UTR extension. The presence of this silencer in the spi 2.3 gene very likely accounts for the lack of basal of transcription in vivo and for induction of the gene during acute inflammation.
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PMID:Transcriptional repression, a novel function for 3' untranslated regions. 764 61

We have examined the involvement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor in the cellular response to GH. Stable Chinese hamster ovary (CHO) cell clones expressing a receptor with tyrosine residues at position 333 and 338 of the receptor substituted for phenylalanine (CHO-GHR1-638 Y333F, Y338F) were generated by cDNA transfection. Compared with the wild type receptor the Y333F,Y338F mutant possessed normal high affinity ligand binding, hormone internalization, and ligand-induced receptor down-regulation. GH activation of mitogen-associated protein kinase was also similar in CHO clones expressing similar wild type and Y333F,Y338F receptor number. However, two GH-regulated cellular events (lipogenesis, and protein synthesis) were deficient in the tyrosine substituted receptor. In contrast, transcriptional regulation by GH (as evidenced by chloramphenicol acetyltransferase cDNA expression driven by the GH-responsive region of the SPI 2.1 gene) was not affected by Y333F,Y338F substitution. Thus we provide the first experimental evidence that specific tyrosine residues of the GH receptor are required for selected cellular responses to GH.
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PMID:Requirement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor for selected GH-stimulated function. 766 93

The 5'-terminal untranslated region (5' UTR) of the uncapped hepatitis A virus (HAV) RNA contains two pyrimidine-rich sequences; one about 20 nucleotides (nt) in length in the vicinity of the AUG initiation codon (nt 706-726), and a longer one (about 40 nt) encompassing nt 100 to 140. The latter includes a 13 nt 'core' sequence (positions 126-138 in the HM175 strain) which is 80% identical to the pyrimidine-rich tract of poliovirus type 1 RNA (Mahoney strain). Representative cDNAs of the entire 5' UTR of HAV RNA were inserted in the intercistronic region of the bi-cistronic plasmid pSV-GH/CAT between the genes coding for the human growth hormone (GH) and bacterial chloramphenicol acetyltransferase (CAT). When COS-7 cells were transfected with these constructs they transiently expressed CAT indicating that the 5' UTR of HAV was efficiently directing internal initiation of translation of the reporter gene. Under similar conditions the 5' UTR of poliovirus type 2 (Lansing strain) was 30% more efficient in directing the expression of the CAT gene. Removal of the 'core' sequence from the 5'-distal pyrimidine-rich stretch extending between nt 117 and 131 in the HAV 5' UTR reduced the CAT activity in the lysates of transfected cells by 40%, whereas point mutations engineered in this segment strongly decreased (80% inhibition) the HAV-driven expression of the reporter gene. Limited mutations systematically introduced in the reiterated (U)UUUCCC motifs of the 5'-distal pyrimidine-rich tract identified two major functional domains extending between nt 100-106 and 113-119. Substitutions in these hexanucleotides abrogated internal initiation of translation, whereas similar changes in the neighbouring domains (nt 107-112 and 120-126) had no effect on the expression of the reporter gene, suggesting that the 5'-most pyrimidine-rich tract is indeed part of the structure(s) recognized by ribosomes and associated factors at initiation of translation and that the hexanucleotides 100-106 and 113-119 constitute an important part of it. Although HAV replicates better at 33 degrees C than at 37 degrees C, incubation of transfected cultures at 33 degrees C delayed the expression and slightly reduced the level of CAT activity in the cell lysates, but the overall effect of the mutations remained unchanged.
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PMID:5' UTR of hepatitis A virus RNA: mutations in the 5'-most pyrimidine-rich tract reduce its ability to direct internal initiation of translation. 773 Aug 3

In previous studies [Hui and de Boer, Proc. Natl. Acad. Sci. USA 84 (1987) (1987) 4762-4766; Hui et al., Methods Enzymol. 153 (1987) 432-452], it was shown that efficient translation of the human growth hormone mRNA (hGH) species having an altered Shine-Dalgarno (SD) sequence, 5'-GUGUG-3', depends on the presence of specialized (spc) ribosomes containing the modified anti-SD (ASD) sequence, 5'-CACAC-3', near the 3' end of their 16S rRNA. In spite of the altered ASD sequence, spc ribosomes were not found to be committed exclusively to the translation of the hGH mRNA; no more than 30% of the total amount of protein synthesized by such ribosomes was hGH. Once we replace the coding sequence of the hGH mRNA with that of chloramphenicol acetyltransferase (CAT), the specificity of spc ribosomes for translation of a single targetted mRNA, relative to the endogenous mRNAs, is greatly enhanced; an estimated 80% of the total amount of protein synthesized by spc ribosomes is CAT. Using the inducible spc ribosome system containing the cat gene, we show that, upon induction, spc ribosomes accumulate in large excess over the number needed for optimal translation of the targetted cat mRNA. Despite the excess, only few spc ribosomes initiate translation on a limited number of endogenous mRNAs. The excessive accumulation of spc ribosomes, which are predominantly present as free 30S subunits, is neither deleterious to the cells, nor does it lead to a feedback inhibition of the synthesis of wild-type ribosomes.
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PMID:Specialized ribosomes: highly specific translation in vivo of a single targetted mRNA species. 775 59

Transient transfection and murine germ line gene transfer analysis was used to determine the regions of DNA necessary to confer the appropriate level and cell specificity of the expression of the gene coding for the murine Clara cell 10-kDa protein, mCC10. To identify the cis-acting elements involved in the regulation of mCC10 gene, different lengths of the 5'-flanking sequence were ligated to the bacterial chloramphenicol acetyltransferase gene for transient transfection to H441 cells (human lung adenocarcinoma cell line). The corresponding sequences were also fused to the human growth hormone gene and transferred to the murine genome for an in vivo analysis of mCC10 promoter activity. The results of the transient transfection analysis identified the region from -166 to -124 of the 5'-flanking region of the mCC10 gene as necessary for the expression of this gene in H441 cells. The transgenic mouse analysis confirmed that the 166 base pairs of 5'-flanking DNA was sufficient to confer cell-specific expression. However, the transgenic mouse analysis also showed that, to achieve the full quantitative level of transgene (human growth hormone) expression, regions between -803 and -166 base pairs of the 5'-flanking sequences are required for maximum expression of mCC10 gene promoter activity.
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PMID:cis-acting elements involved in the regulation of mouse Clara cell-specific 10-kDa protein gene. In vitro and in vivo analysis. 785 38

Growth hormone activates gene transcription of the serine protease inhibitors (SPI) 2.1 and 2.2 by an unknown mechanism. In order to define the promoter regions responsible for this effect and to characterize the transcription factors involved, we have performed gel electrophoresis mobility shift assays on nuclear extracts from cell lines transfected with growth hormone receptor cDNA. We have identified a 9-base pair DNA element, the SPI-GLE 1, which forms a complex with nuclear proteins following activation by growth hormone and which, when placed upstream of a minimal thymidine kinase promoter, drives chloramphenicol acetyltransferase expression in a growth hormone-dependent fashion. This element is similar to those from several genes regulated by other cytokines including interferon. The growth hormone-induced complexes formed were dependent on tyrosine phosphorylation but did not contain the interferon-gamma-activated transcription factor Stat 91. Competition studies with oligonucleotides similar to the SPI-GLE 1 reveal the sequence of a consensus element that specifically binds growth hormone-regulated nuclear proteins.
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PMID:Growth hormone specifically regulates serine protease inhibitor gene transcription via gamma-activated sequence-like DNA elements. 792 35

An expression vector designed to express a long (approximately 1 kb) RNA containing multiple ribozyme domains was cotransfected into mammalian cells with a plasmid encoding, as target, messenger RNA for chloramphenicol acetyltransferase (CAT). In comparative studies the multimeric ribozyme construct proved to be significantly more effective at suppressing CAT expression than either the corresponding antisense RNA, or a transcript carrying a single ribozyme domain. Suppression of gene activity was apparently specific because expression of an independently expressed gene for human growth hormone was unaffected. The profile of CAT RNA extracted from transfected cells was consistent with RNA cleavage.
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PMID:Multiple domains in a ribozyme construct confer increased suppressive activity in monkey cells. 795 Mar 4


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