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)

Viruses that establish persistent infections may show selective and unique effects on the host's transcriptional machinery. Lymphocytic choriomeningitis virus (LCMV), a noncytolytic virus, can persistently infect a rat pituitary cell line. Although the infected cells remain free of structural damage, virus markedly interferes with growth hormone (GH) but only minimally interferes with prolactin transcription. The study of GH promoter-chloramphenicol acetyltransferase-transfected cells and GH promoter deletion mutants demonstrates that the viral effect is at the level of GH promoter and is due to interference with GH transactivator factor GHF1 (Pit1). Treatment of LCMV-infected cells with the antiviral agent ribavirin cures the infection and restores normal GH mRNA levels. These results illustrate a molecular mechanism by which a virus infection can disrupt synthesis of a cell's differentiated product without perturbing vital cellular functions.
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PMID:Selective disruption of growth hormone transcription machinery by viral infection. 140 23

An insulin response element (IRE) has been identified in the prolactin gene using chimeric plasmids in which prolactin promoter DNA directs expression of the bacterial chloramphenicol acetyltransferase gene. A series of 5'-deletion constructs starting between positions -173 and -106 and extending through position +75 of the prolactin gene were all stimulated greater than 10-fold by physiological concentrations of insulin in rat pituitary tumor GH4 cells. However, insulin did not stimulate constructs starting at positions -96 and -46, suggesting that the IRE of the prolactin gene may be located in region -106/-96. Insulin stimulation of prolactin-chloramphenicol acetyltransferase constructs requires cotransfection with a human insulin receptor expression vector. Estimation of insulin receptor levels by beta-subunit phosphorylation indicates that receptor levels are increased approximately 50-fold following transfection with the human insulin receptor expression vector. This requirement for cotransfection suggests that the endogenous receptor levels may not be adequate to couple the response of transfected genes to insulin. Gel mobility shift experiments reveal a nuclear factor from GH4 cells that specifically associates with prolactin DNA fragment -106/-87. The amount or binding activity of this factor is increased following insulin treatment of cells. The concordance between functional and binding analyses of the prolactin promoter confirms the presence of an IRE in region -106/-87. The insulin-sensitive DNA-binding factor may mediate effects of insulin on prolactin gene transcription.
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PMID:An element in the prolactin promoter mediates the stimulatory effect of insulin on transcription of the prolactin gene. 164 45

To identify the cis-acting elements responsible for cAMP stimulation of human prolactin (hPRL) promoter activity, pituitary GC cells were transfected with 5'-deleted hPRL promoters fused to the chloramphenicol acetyltransferase reporter gene. The proximal regulatory region (coordinates -250 to -42) was sufficient to confer strong cAMP stimulation (+/- 25 fold). Further 5' and 3' deletions performed within this proximal region demonstrated that two types of cis-acting elements are involved in the cAMP regulation: (i) the binding sites of the pituitary-specific factor Pit-1, and (ii) the sequence between coordinates -115 and -85 (named fragment A), which contains a TGACG motif. We show by gel-shift and Southwestern experiments that fragment A binds Pit-1 monomer and also a ubiquitous factor that is neither cAMP-responsive element-binding protein nor activator protein-1. Strong cAMP induction was observed when fragment A was juxtaposed to a Pit-1 binding site. That Pit-1 plays an important role was supported further by the finding that the hPRL proximal region conferred cAMP regulation when linked to the herpes simplex virus thymidine kinase promoter only in pituitary GC cells and not in other heterologous cells, which do not express Pit-1. Furthermore, we observed that concatenated Pit-1 binding sites were able to confer cAMP responsiveness to the thymidine kinase promoter in GC cells.
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PMID:Transcriptional induction of the human prolactin gene by cAMP requires two cis-acting elements and at least the pituitary-specific factor Pit-1. 165 38

Hypophyseal portal dopamine is a major negative regulator of pituitary prolactin (PRL) production. Dopamine has been reported to repress PRL gene transcription in pituitary cells. To facilitate further study of the effect of dopamine on PRL gene activation, we introduced PRL promoter and D2 receptor (D2R) constructs into GH3 cells. Since two D2R isoforms (termed D2S and D2L) have been cloned previously, we first determined which isoform(s) is present in the lactotroph by measuring the level of each mRNA species in rat prolactinoma. mRNA for each D2R isoform was found to be present, with the D2L mRNA in great (c. 6-fold) excess. Because the lactotroph contains both isoforms, the effect of each on the PRL promoter was investigated. The cDNA for each receptor isoform was synthesized by polymerase chain reaction, and cloned into an RSV-based expression vector. GH3 cells were then transiently co-transfected with either of the resulting RSV-D2R constructs plus a PRL-chloramphenicol acetyltransferase (CAT) construct containing the first 1957 base-pairs of PRL gene 5'-flanking DNA. The cells were then incubated 48 h plus or minus the dopamine agonist ergocryptine (ECR). In the presence of either RSV-D2R isoform, ECR yielded a 4-5-fold decrease in CAT activity, an effect not seen in the absence of the RSV-D2R. The promoter specificity of this effect was demonstrated by the inability of ECR to regulate expression of a control RSV-CAT construct. The PRL promoter repression mediated by each receptor isoform had appropriate pharmacology: the specific D2R agonist, quinpirole, yielded results similar to ECR, and the ECR repression was reversed by the dopamine antagonist spiperone.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Either isoform of the dopamine D2 receptor can mediate dopaminergic repression of the rat prolactin promoter. 183 94

The biological activities of long and short forms of the prolactin receptor have been compared. These two receptors expressed in mammalian cells were shown to bind prolactin with equal high affinity. The ability of these different forms to transduce the hormonal message was estimated by their capacity to stimulate transcription by using the promoter of a milk protein gene fused to the chloramphenicol acetyltransferase (CAT) coding sequence. Experiments were performed in serum-free conditions to avoid the effect of lactogenic factors present in serum. An approximately 17-fold induction of CAT activity was obtained in the presence of prolactin when the long form of the prolactin receptor was expressed, whereas no induction was observed when the short form was expressed. The present results clearly establish that only the long form of the prolactin receptor is involved in milk protein gene transcription.
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PMID:Comparison of long and short forms of the prolactin receptor on prolactin-induced milk protein gene transcription. 199 73

Two alternating purine-pyrimidine sequences of the d(TG)n.d(CA)n-type (170bp and 60 bp in length) lie upstream of the rat prolactin (rPRL) gene. Conformational studies of plasmids containing these sequences indicate that both form left-handed (Z) DNA, with transitions initiating at superhelical densities of -0.041 and -0.044 respectively. These alternating purine-pyrimidine (APP) sequences are hypersensitive to cleavage with S1 nuclease both at the boundaries and within these APP repeats, where there is a loss in APP alternation. We have investigated the function of one of these Z-DNA sequences in the regulation of rPRL transcription, by linking regions of the 5' flanking sequence of the rPRL gene to a reporter gene encoding chloramphenicol acetyltransferase (CAT), and transferring these plasmids into GH3 pituitary tumour cell lines. The major conclusion from these studies is that the 170bp repeat exerts a negative effect on the transcription of the rPRL gene, and also down-regulates the expression of the fusion gene pRSVcat when cloned 50bp upstream of the Rous sarcoma virus promoter. However, despite its proximity to an estrogen response element in prolactin, this sequence does not affect the responsiveness of the rPRL gene to estrogen.
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PMID:d(TG)n.d(CA)n sequences upstream of the rat prolactin gene form Z-DNA and inhibit gene transcription. 215 81

An efficient electroporation procedure was established for the genetic transformation of two clonal strains of hormone producing rat pituitary cells (GH12C1 and GH3). We used the bacterial chloramphenicol acetyltransferase (CAT) gene as reporter gene to determine optimal conditions for electroporation. The conditions found to be optimal, measured as expression of the highest CAT activity, were 240-300 V and a DNA concentration of 30-60 micrograms/ml in sucrose buffer. Cell viability was then about 50 per cent. Maximum CAT activity was seen 24 hours after electroporation. The electroporation procedure, in the presence or absence of DNA, caused a transient decrease in endogenous growth hormone (GH) and prolactin (PRL) production.
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PMID:Electroporation of rat pituitary (GH) cell lines: optimal parameters and effects on endogenous hormone production. 222 25

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express beta-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete beta-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine beta-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency (greater than 150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the beta-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous beta-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.
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PMID:Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells. 225 Dec 52

The prolactin (PRL) gene in clonal strains of rat pituitary tumor cells in culture (GH cells) exhibit several regulatory responses, similar to the ones observed in rat pituitary gland. A comparative analysis of regulation of PRL gene expression in PRL-producing (PRL+) and PRL-nonproducing (PRL-) GH cells was conducted by monitoring the PRL promoter driven transient expression of chloramphenicol acetyltransferase (CAT) gene in GH4C1 (PRL+) and GH12C1 (PRL-) cells. The PRL promoter activity was drastically inhibited only in PRL-nonproducing cells (PRL-) and not in PRL producing cells (PRL+) when a 80-base pair (bp) DNA sequence from 5'-flanking region of PRL gene (located between -330 and -250 bp) was included in the PRL-CAT fusion gene constructs. Furthermore, a DNA/protein interaction involving this 80-bp DNA sequence and a 60-kDa nuclear protein was detected only in PRL- cells but not in PRL+ GH cells. These results suggested that the strain-specific suppression of PRL gene in PRL-, GH12C1 cells was mediated via interaction of a cis-acting negative regulatory element with a negative regulatory trans-acting factor in these cells. The negative regulatory element within the AT-rich 80-bp DNA sequence was mapped immediately adjacent to the site of interaction of trans-activators of PRL gene.
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PMID:cis-acting negative regulatory element of prolactin gene. 231 63

We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.
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PMID:Regulatory elements controlling pituitary-specific expression of the human prolactin gene. 2388 22


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