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)

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.
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PMID:Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene. 193 51

DNA sequences in the 5'-flanking region of rat and bovine oxytocin genes were examined for their capacity to confer estrogen responsiveness to their homologous promoters. In contrast to the 5'-flanking region of the rat oxytocin gene, upstream promoter sequences up to 3200 bp of the bovine gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene which were transfected in estrogen receptor expressing MCF-7 cells did not respond to estrogen. Testing 5'-deletion mutants of the rat upstream region linked to the luciferase gene in P19 embryocarcinoma cells co-transfected with an estrogen receptor expression plasmid showed that two regions each associated with approximately 15-fold stimulation of promoter activity were located between nucleotides -172 and -149 and between -148 and +16 in the rat gene. The former region contains the imperfect palindrome GGTGACCTTGACC which differs in one nucleotide from the estrogen response element (ERE) consensus. It is concluded that the corresponding motive CATAACCTTGACC of the bovine gene is not a functional ERE. Thus, the estrogen responsiveness of oxytocin genes is species-dependent.
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PMID:Comparison of the estrogen responsiveness of the rat and bovine oxytocin gene promoters. 199 97

We showed previously that a gene construction that consisted of 5.2 kb of 5' flanking sequence, the first exon, and part of the first intron of the human gene encoding vasoactive intestinal peptide (VIP) fused to the reporter gene chloramphenicol acetyltransferase (CAT) fully mimicked the diverse behavior of the endogenous VIP gene when transfected into subclones of the human neuroblastoma cell line SK-N-SH (Waschek et al., 1988). To determine if the same sequences were sufficient to target expression of a reporter to VIP-producing tissues in the mouse, we initiated a pilot study in which we generated four transgenic mice or mouse lines that contained the VIPCAT fusion gene. Detectable levels of CAT were found in the ileum of either founder or offspring of each of the transgenic mouse lines. In all other tissues tested, CAT activity was either below the level of detection or the transgene was not expressed, with the exception of one mouse in which ectopic expression in the cerebellum was observed. The results indicate that the VIP sequences utilized were sufficient to direct expression of the transgene to the intestine, but not necessarily to other sites of VIP expression. To investigate what specific DNA sequences might confer VIP expression in the intestine and other sites, we analyzed further the VIP gene in SK-N-SH subclones using VIP/luciferase fusion gene constructions. A 0.6 kb DNA fragment located between 4.0 kb and 4.6 kb upstream from the VIP transcriptional start site was found to impart a high level of expression in one subclone and an increased degree of phorbol ester induction in another. These and other data indicate that multiple transcriptional elements control VIP expression in neuroblastoma cells and are candidates as mediators of VIP gene expression in the intact animal.
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PMID:Expression of a chimeric VIP gene is targeted to the intestine in transgenic mice. 207 11

Using Tn4431, a transposon that allows transcriptional fusions to a promoterless luciferase (lux) operon, we have isolated a nonpathogenic mutant of Xanthomonas campestris pv. campestris, i.e., JS111, that does not incite any of the black rot symptoms on all tested cruciferous host plants (J. J. Shaw, L. G. Settles, and C. I. Kado, Mol. Plant Microbe Interact. 1:39-45, 1988). In the study reported here, we determined that in contrast to the wild-type strain, JS111 is unable to induce a hypersensitive necrotic response on nonhost plants such as datura, tomato, and cucumber, suggesting that JS111 is a nonpathogenic, nonhypersensitive Hrp mutant. JS111 displayed culture growth rates, exopolysaccharide production, and protease, pectate lysase, cellulase, amylase, and phosphatase activities comparable to those of the wild-type strain. However, the growth of JS111 in host leaves was markedly attenuated. Coinoculation of JS111 with the wild-type strain in cauliflower or radish leaves rescued the growth deficiency of the mutant to normal levels. The locus mutated in JS111 was cloned and named hrpXc, and transcriptional and genetic complementation analyses of the hrpXc locus were conducted. The regulation of hrpXc expression was also investigated in vitro and in planta, using fusions to a lux or chloramphenicol acetyltransferase reporter gene. The hrpXc gene was found to be strongly induced in radish leaves. This is the first report and analysis of a hrp locus from a Xanthomonas species.
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PMID:A plant-inducible gene of Xanthomonas campestris pv. campestris encodes an exocellular component required for growth in the host and hypersensitivity on nonhosts. 216 73

Since human immunodeficiency virus (HIV) nef has been suggested to exert regulatory effects on HIV long terminal repeat (LTR) activity, we transiently transfected HIV LTR chloramphenicol acetyltransferase or luciferase expression vectors into a human astrocytoma clone (U-373nef) that constitutively expresses the HIV nef gene. In these cells, basal HIV LTR activity, as well as tumor necrosis factor-induced or tat-driven activity, was similar to that in control cells. Lack of any detectable effect of HIV nef on LTR activity was not the result of mutations in integrated nef DNA, as was shown by polymerase chain reaction. These data suggest that the role of nef in HIV genome transcription does not necessarily involve a direct influence on HIV LTR activity.
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PMID:Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity. 218 77

The small marine ostracod crustacean, Vargula hilgendorfii, produces a bright blue luminous secretion which is ejected into seawater. The luminescence is due to a simple enzyme-catalyzed reaction involving only luciferase, luciferin (substrate), and molecular oxygen. Thus, V. hilgendorfii luciferase (VL) should be useful as a reporter enzyme in studies of gene expression in mammalian cells. Expression plasmids consisting of VL cDNA (vl) linked to the promoters simian virus 40 early region, Rous sarcoma virus long terminal repeat, human elongation factor, or mouse granulocyte colony-stimulating factor were introduced into a series of mammalian cell lines. Following transfection, VL activities in cell extracts and culture media were determined by a rapid light emission assay with V. hilgendorfii luciferin. Parallel experiments were carried out with the chloramphenicol acetyltransferase (CAT)-encoding gene. In all cell lines tested, VL was secreted, allowing the reporter activity to be determined directly from a small aliquot of the culture medium. The results indicate that the secreted VL enzyme is superior to CAT, firefly luciferase, and bacterial luciferase as a convenient and versatile indicator of gene expression in mammalian cells.
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PMID:Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells. 226 35

Typical of other housekeeping genes, the promoter for the human hypoxanthine phosphoribosyl transferase-encoding gene (HPRT) is G + C-rich, lacks a TATA box and has multiple transcription start points. To test the hypothesis that these features may result in relaxed control over the direction of transcription, we examined the effect of orientation on the ability of the HPRT promoter to control expression of the following reporter genes in transfected cells: luc (firefly luciferase), cat (bacterial chloramphenicol acetyltransferase) and neo (neomycin resistance). A 376-bp fragment containing the HPRT promoter efficiently expressed the luc gene irrespective of orientation, and the 5' ends of luciferase RNA produced in cells transfected with inverted promoter constructs mapped to within the HPRT promoter, indicating that the HPRT promoter has bidirectional activity. However, in the presence of two divergently-flanking reporter genes expression from the inverted HPRT promoter was only 10-20% compared to the noninverted orientation. Furthermore, the inverted HPRT promoter expressed cat less well than luc, and was unable to express neo sufficiently well to produce any colonies under appropriate selection conditions. Attempts to detect endogenous divergent HPRT transcripts were unsuccessful. The promoter of another housekeeping gene, encoding 3-phosphoglycerate kinase (PGK), expressed moderate levels of cat (40%) but not luc (less than 5%) in the inverted orientation. By comparison, two TATA-box containing promoters functioned extremely poorly when inverted. This study indicates that two plasmid-borne housekeeping promoters have at least a limited potential for bidirectional activity, but the functional significance of this is unclear if the corresponding endogenous housekeeping promoters express divergent transcripts at similarly low levels. The poor activity of the HPRT and PGK promoters in the inverted orientation suggests that there is a mechanism which influences the direction of transcription from these promoters.
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PMID:Limited bidirectional activity of two housekeeping gene promoters: human HPRT and PGK. 234 94

12-O-Tetradecanoylphorbol-13-acetate (TPA) activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene in the wild type PC-12 cells but not in the variant PC-12 cells that originated from the wild type cells. Transfection of the c-Ha-rasval12 complementary DNA (cDNA) or addition of dibutyryl cAMP to the wild type PC-12 cells as well as to the variant PC-12 cells activated the c-fos gene enhancer. Prolonged treatment of the wild type PC-12 cells with phorbol-12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-Ha-rasval12 cDNA still stimulated the c-fos gene enhancer to the same extent as induced in the control cells. Transfection of the c-Ha-rasval12 cDNA or addition of TPA to the wild type PC-12 cells stimulated the serum-response element but not the cAMP-response element. Dibutyryl cAMP stimulated both the serum-response element and the cAMP-response element in the wild type PC-12 cells. These results indicate that the c-Ha-rasval12 protein activates the serum-response element, but not the cAMP-response element in the c-fos gene enhancer, and that the signal pathway from the c-Ha-rasval12 protein to the c-fos serum-response element is independent of protein kinase C and cAMP-dependent protein kinase.
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PMID:Activation of the c-fos serum-response element by the activated c-Ha-ras protein in a manner independent of protein kinase C and cAMP-dependent protein kinase. 240 11

Transfection of the cDNA encoding the activated c-raf-1 protein or addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) or dibutyryl cAMP to NIH/3T3 cells activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene. Prolonged treatment of NIH/3T3 cells with phorbol 12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, addition of TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-raf-1 cDNA or addition of dibutyryl cAMP still stimulated the c-fos gene enhancer to the same extent as those induced in the control cells. Transfection of the c-raf-1 cDNA or addition of TPA to NIH/3T3 cells stimulated the serum response element and TPA response element but not the cAMP response element. In contrast, addition of dibutyryl cAMP to NIH/3T3 cells stimulated the cAMP response element but not the serum response element or TPA response element. These results indicate that the activated c-raf-1 protein stimulates the serum response element and TPA response element in a manner independent of protein kinase C and cAMP-dependent protein kinase. Since the c-fos gene enhancer has been shown to contain the serum response element and cAMP response element, it is most likely that the c-raf-1 protein is involved in the regulation of c-fos gene expression through the serum response element.
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PMID:Activation of the serum response element and 12-O-tetradecanoylphorbol-13-acetate response element by the activated c-raf-1 protein in a manner independent of protein kinase C. 255 85

Short catalytic RNAs possessing specific endoribonuclease activity (ribozymes) have recently been designed that can potentially shear any chosen target RNA in trans at a specific site. Here, engineered ribozymes targeted against chloramphenicol acetyltransferase (CAT), derived from Tn9, have been cloned into a mammalian expression vector and tested in transient transfection experiments for their effects on CAT expression in monkey (COS1) cells. The ribozymes contained the catalytic domain of the satellite RNA from tobacco ringspot virus and were targeted to three sites in the CAT mRNA by flanking antisense sequences. These ribozymes, which were previously shown to accurately cleave CAT message in vitro, were cloned into a replicating plasmid vector under the control of the highly active simian virus 40 early promoter. The ribozyme gene sequence was incorporated into the 3' untranslated region of the gene for firefly luciferase as it was ineffective when expressed as a short RNA. Each ribozyme construction gave a similar level of suppression of CAT activity when the target was transcribed from the herpes virus thymidine kinase promoter. One of the three (ribozyme 2) was chosen for further study and tested after it had been modified by the addition of extra flanking bases. The reporter gene for luciferase was used to monitor ribozyme level and to function as a specificity control, and the human growth hormone gene was cotransfected as an independent reporter for specificity of the ribozyme against the intended target CAT. At high (approximately 1000-fold) molar excess this ribozyme was demonstrated to consistently and specifically suppress CAT expression (up to approximately 60%) in COS1 cells relative both to a plasmid clone with the ribozyme inserted in the reversed (inactive) orientation and to a control corresponding to the relevant 26-nucleotide antisense segment of CAT.
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PMID:Specific gene suppression by engineered ribozymes in monkey cells. 255 2


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