An Improved CAT Assay for Promoter Analysis in Either Transgenic Mice or Tissue Culture Cells

Pothier, F.; Ouellet, Mario; Julien, Jean‐Pierre; Guérin, Sylvain L.

doi:10.1089/dna.1992.11.83

Cited by 160 publications

(78 citation statements)

References 26 publications

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“…(39). Drosophila Schneider cells were transfected according to the calcium phosphate precipitation procedure (36,40) at a density of 1 ϫ 10 6 cells per 60-mm culture plate.…”

Section: Methodsmentioning

confidence: 99%

“…Levels of CAT activity for all transfected cells were determined as described (40) and normalized to the amount of hGH secreted into the culture media and assayed using a kit for quantitative measurement of hGH (Immunocorp, Montréal, Québec, Canada). Because the metallothionein-I promoter, which directs expression of hGH from the pXGH5 plasmid, proved to be highly inefficient in Drosophila cells, CAT activities from transfected Schneider cells were normalized to the amount of ␤-galactosidase encoded by the plasmid pAC5/V5-His/LacZ and cotransfected along with the CAT recombinant constructs.…”

Section: Methodsmentioning

confidence: 99%

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Expression of the α5 Integrin Subunit Gene Promoter Is Positively Regulated by the Extracellular Matrix Component Fibronectin through the Transcription Factor Sp1 in Corneal Epithelial Cells in Vitro

Larouche

Leclerc

Salesse³

et al. 2000

Journal of Biological Chemistry

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The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin ␣ 5 ␤ 1 . In this work, we determined whether the activity directed by the ␣ 5 gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/␣ 5 promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediating FN responsiveness was shown to bear a perfect inverted repeat that we designated the fibronectin-responsive element (FRE). Analyses in electrophoretic mobility shift assays provided evidence that Sp1 is the predominant transcription factor binding the FRE. Its DNA binding affinity was found to be increased when RCEC are grown on FN-coated dishes. The addition of the MEK kinase inhibitor PD98059 abolished FN responsiveness suggesting that alteration in the state of phosphorylation of Sp1 likely accounts for its increased binding to the ␣ 5 FRE. The FRE also proved sufficient to confer FN responsiveness to an otherwise unresponsive heterologous promoter. However, site-directed mutagenesis indicated that only the 3 half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its ␣ 5 ␤ 1 integrin activates a signal transduction pathway that results in the transcriptional activation of the ␣ 5 gene likely through altering the phosphorylation state of Sp1.Corneal wounds account for a substantial proportion of all visual disabilities and medical consultations for ocular problems in North America. They can be superficial with damage limited to the epithelium or associated with a deeper involvement of the epithelial basement membrane and of the stromal lamella. Severe recurrent and persistent corneal wounds are most commonly secondary to ocular diseases and damage such as recurrent erosion, mild chemical burns, superficial herpetic infections, neuroparalytic cornea, autoimmune diseases, and stromal ulcerations due to viral or bacterial infections or to severe burns (1). Despite currently available treatments, many of these corneal wounds persist for weeks and months or else recur frequently and can progress to corneal perforation.Tissue repair requires cell migration, proliferation, and adhesion. Cell adhesion and migration in turn require extracellular matrix (ECM) 1 synthesis and assembly. ECM is a complex, cross-linked structure of proteins and polysaccharides. It organizes the geometry of normal tissues. Fibronectin (FN) is an ECM adhesion protein identified as a potential wound healing agent because of its cell attachment, migration, differentiation, and orientation properties (for a review see Refs. 2-4). In the unwounded rat eye, FN is observed by immunohistological staining at the level of the corneal epithelium basement membrane (5-7). Shortly after corneal injury, the basal cells that border the injured area and stromal keratocytes start producing massive amounts of FN (5,[8]...

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“…(39). Drosophila Schneider cells were transfected according to the calcium phosphate precipitation procedure (36,40) at a density of 1 ϫ 10 6 cells per 60-mm culture plate.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Expression of the α5 Integrin Subunit Gene Promoter Is Positively Regulated by the Extracellular Matrix Component Fibronectin through the Transcription Factor Sp1 in Corneal Epithelial Cells in Vitro

Larouche

Leclerc

Salesse³

et al. 2000

Journal of Biological Chemistry

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“…Phenol red-free RPMI-1640 medium (500 ml) supplemented with 15% charcoal-treated FBS was added 4 to 8 h after transfection, at this time ligand was also added. The cells were harvested 40 h after treatment and CAT-assays were performed as described (Pothier et al, 1992). The CAT activities were normalized to b-galactosidase activity and induction factors were calculated as the ratio of CAT activity of ligand-stimulated cells to that of mock-induced controls.…”

Section: Transfection and Reporter Gene Assaymentioning

confidence: 99%

Differential apoptotic response of human melanoma cells to 1α,25-dihydroxyvitamin D3 and its analogues

et al. 1998

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Pleiotropic actions of the biologically active form of vitamin D 3 , 1a,25-dihydroxyvitamin D 3 (VD), include antiproliferative effects in both normal human melanocytes and malignant melanoma cell lines. In this study the actions of VD and its low calcemic analogues EB1089 and CB1093, have been examined in two human melanoma cell lines MeWo and WM1341. Both cell lines express similar amounts of vitamin D receptor mRNA and show functional gene regulatory effects in response to VD and its analogues. VD, EB1089 and CB1093 induced apoptosis only in WM1341 cells and not in MeWo cells, even though both cell lines responded well to etoposide, a strong inducer of apoptosis. Additionally, these results were confirmed by analysis of cell morphology. Interestingly in WM1341 cells, CB1093 was found to be more potent in inducing apoptosis than EB1089 and the natural hormone. Moreover, CB1093 appeared to induce apoptosis at a relatively low concentration of 0.1 nM, whereas greater than tenfold higher concentrations of VD and EB1089 were needed to obtain comparable effects. These observations highlight CB1093 as a promising drug for a future treatment against specific types of melanoma. -23yne-24a,26a, 27a-trihomo-1a,25-dihydroxyvitamin D 3 ; CAT, chloramphenicol acetyl transferase; DR3, direct repeat spaced by 3 nucleotides; EB1089, 22,24-diene-24a,26a,27a-trihomo-1a,25-dihydroxyvitamin D 3 ; IP9, inverted palindrome spaced by 9 nucleotides; TNFa, tumor necrosis factor alpha; VD, 1a,25-dihydroxyvitamin D 3 ; VDR, 1a,25-dihydroxyvitamin D 3 receptor; VDRE; VD response element

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“…Approximately 2 ϫ 10 6 viable protoplasts were transfected with an electroporator (Bio-Rad) as described (24) CAT Assay-Protoplast lysates were prepared in CAT Buffer A (25) as described previously (21). Protein in these lysates was quantified with Bradford reagent (Bio-Rad), and 1-20 g was used for determining CAT activity by a radioanalytic method (25). In this assay, [ 14 C]chloramphenicol was used as substrate and was separated from mono-and diacetylated reaction products by thin-layer chromatography (TLC) with 5% methanol and 95% chloroform.…”

Section: Construction Of Effector and Reportermentioning

confidence: 99%

Auxin-induced Stress Potentiates trans-activation by a Conserved Plant Basic/Leucine-zipper Factor

Pascuzzi

Hamilton

Bodily

et al. 1998

Journal of Biological Chemistry

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The promoter element activation sequence-1 (as-1) confers tissue-specific and signal-responsive transcription in plants. Hormone and chemical stress cues are thought to activate as-1-dependent transcription through specific basic/leucine-zipper proteins, termed TGA factors, that bind this element. We report here that a highly conserved TGA factor of tobacco, TGA1a, can selectively activate transcription in response to micromolar concentrations of auxin hormones or their analogs. This induction is chemically specific, as a range of other compounds tested at similar concentrations had little or no effect. Auxin was found to augment the transactivation potential of TGA1a through carboxyl-terminal residues. The amino-terminal domain of TGA1a, by gain-of-function assays, was found to both constitutively activate transcription and maximize the response to auxin. Further evidence indicates that the trans-activation potential of this domain in TGA1a is repressed, under basal conditions, by carboxyl-terminal residues. Because TGA1a and endogenous TGA factors are stimulated by auxin only at concentrations that inhibited cell growth, this response is likely to involve chemical stress.The expression of nuclear genes in response to cellular and environmental cues is largely regulated by trans-acting factors and their cognate cis-elements. In plants, auxin hormones (e.g., indole-3-acetic acid) promote growth and affect a number of cellular processes. Auxins are believed to mediate at least some of their effect by enhancing the expression of specific genes through target cis-elements (1, 2). The prototype for one class of auxin-responsive element is activation sequence-1 (as-1), 1 which was first identified by Lam et al. (3) in the cauliflower mosaic virus (CaMV) 35 S promoter. Homologs of as-1 (e.g., ocs and nos), moreover, occur in the promoters of plant-transforming T-DNA genes of agrobacteria (4 -6). Apart from regulating these genes in plants, a broader role for as-1 elements has been suggested by their functional presence in plant glutathione S-transferase genes (7-9). For example, auxin-responsive expression of at least one member of the tobacco GST gene family, GNT35, is mediated by as-1 (8, 9). Unlike other auxin-responsive elements, as-1 requires physiologically high (i.e. micromolar) concentrations of auxins to activate transcription, suggesting that this response may involve chemical stress. This view is further supported by the observation that biologically inactive analogs of auxins are also strong inducers of as-1-dependent activity.Multiple TGA factors that bind selectively to as-1 and related motifs have been cloned from several plant species, and some or all of these factors are likely to mediate as-1-dependent transcription. Based on their amino acid homology, at least three subclasses of TGA factors occur in plants, with multiple members in each class. Heterodimerization between these factors may generate new combinations with potentially distinct functional properties (10). Posttranscriptional regulation ...

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An Improved CAT Assay for Promoter Analysis in Either Transgenic Mice or Tissue Culture Cells

Cited by 160 publications

References 26 publications

Expression of the α5 Integrin Subunit Gene Promoter Is Positively Regulated by the Extracellular Matrix Component Fibronectin through the Transcription Factor Sp1 in Corneal Epithelial Cells in Vitro

Expression of the α5 Integrin Subunit Gene Promoter Is Positively Regulated by the Extracellular Matrix Component Fibronectin through the Transcription Factor Sp1 in Corneal Epithelial Cells in Vitro

Differential apoptotic response of human melanoma cells to 1α,25-dihydroxyvitamin D3 and its analogues

Auxin-induced Stress Potentiates trans-activation by a Conserved Plant Basic/Leucine-zipper Factor

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