Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:APRD00631 (Gel)
 14,881 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enzymatic activity of bacterial luciferase from Beneckea harveyi (a heterodimer, Mr = approximately 79,000) is rapidly lost upon treatment with trypsin or chymotrypsin. Under nondenaturing conditions, the proteolytically inactivated molecule has the same apparent molecular weight as the native enzyme, and appears to be relatively stable to further proteolytic degradation. Gel electrophoresis in sodium dodecyl sulfate of the products of this digestion shows that only the alpha subunit is degraded during the time of these experiments, and its rate of loss is the same as the rate of loss of light-producing activity. The action of either protease produces a species with mobility indicative of a molecular weight of about 28,000 and smaller fragments, and an unaltered beta subunit.
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PMID:Proteolytic inactivation of the luciferase from the luminous marine bacterium Beneckea harveyi. 30 51

Endocrine factors involved in the transcriptional regulation of the oxytocin (OT) gene were investigated in heterologous expression systems. Plasmids having a 5'-flanking region of the rat OT gene (-363/+16) or the human OT gene (-382/+41) cloned in front of the firefly luciferase gene were co-transfected with an expression vector for the rat thyroid hormone receptor alpha in P19 embryonal carcinoma (EC) cells. Thyroid hormone (T3) stimulated the activity of the rat and human OT promoters about 10-fold. In MCF-7 breast tumor cells transfected with the human OT promoter-luciferase fusion gene, T3 stimulation through endogenous thyroid hormone receptors was about 5-fold. Co-transfection experiments in P19EC cells using 5' deletion mutants of the rat OT gene showed that thyroid hormone responsiveness was located in two regions, one located between nucleotides -195 and -172, the other between nucleotides -172 and -148. Each region accounted for about 3-fold T3 stimulation. Gel retardation analysis using extracts from HeLa cells over-producing the c-erbA/TR alpha protein showed specific binding to the -172/-148 element, while no binding occurred on the -195/-172 element. The -172/-148 element which contains the imperfect estrogen response element, GGTGACCTTGACC, has inverted as well as direct repeats of the TGACC motif. Mutagenesis of TGACC motifs separately reduced thyroid hormone responsiveness by about 50%. However, simultaneous mutation of two TGACC motifs abolished the responsiveness to T3 completely. There was no cooperativity between the activated thyroid hormone and estrogen receptors in transfected MCF-7 cells nor in thyroid hormone receptor and estrogen receptor co-transfected P19EC cells. Negative interactions between these two receptors were observed and gel retardation assays showed interaction between the two receptors proteins. It was shown in an in vivo experiment that treatment of rats with thyroid hormone increased hypothalamic OT mRNA levels, the pituitary OT content, as well as OT levels in blood. The results reveal thyroid hormone as a physiological regulator of OT gene expression, which stimulates OT promoter activity directly through interaction with a thyroid hormone-response element in the OT gene.
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PMID:Thyroid hormone regulates the oxytocin gene. 137 Dec 78

Ciprofibrate, a hypolipidemic drug that acts as a peroxisome proliferator, induces the transcription of genes encoding peroxisomal beta-oxidation enzymes. To identify cis-acting promoter elements involved in this induction, 5.8 kilobase pairs of promoter sequence from the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (EC 4.2.1.17/EC 1.1.1.35) was inserted upstream of a luciferase reporter gene. Transfection of this expression vector into rat hepatoma H4IIEC3 cells in the presence of ciprofibrate resulted in a 5- to 10-fold, cell type-specific increase in luciferase activity as compared to cells transfected in the absence of drug. A peroxisome proliferator-responsive element (PPRE) was localized to a 196-nucleotide region centered at position -2943 from the transcription start site. This PPRE conferred ciprofibrate responsiveness on a heterologous promoter and functioned independently of orientation or position. Gel retardation analysis with nuclear extracts demonstrated that ciprofibrate-treated or untreated H4IIEC3 cells, but not HeLa cells or monkey kidney cells, contained sequence-specific DNA binding factors that interact with the PPRE. These results have implications for understanding the mechanisms of coordinated transcriptional induction of genes encoding peroxisomal proteins by hypolipidemic agents and other peroxisome proliferators.
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PMID:Identification of a peroxisome proliferator-responsive element upstream of the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. 150 66

Previously, 5' deletion analysis revealed three important upstream regions within the regulatory region of the cAMP-induced, prespore gene SP60 of D. discoidium, each of which contains a CA-rich sequence element (CAE: consensus CACACAYYYCACACAAA/T). In this study, we have made site-directed mutations within these CAEs and examined their effect on reporter gene activity (luciferase or lacZ). Point mutations within or deletion of the distal CAE (CAE-1), middle CAE (CAE-2) or proximal CAE (CAE-3) result in substantial decreases in promoter activity at 18 h of development or in response to cAMP. lacZ fusions made with the CAE mutant promoters produced novel beta-gal staining patterns that suggest the presence of one or more morphogen gradients within the prespore zone of the slug and indicate that the CAEs are also important in regulating the spatial patterning of SP60 expression in the multicellular aggregate. Gel mobility shift assays were used to identify activities from crude nuclear extracts that bind oligonucleotides containing the CAEs. One of the binding activities is not observed in extracts from vegetative cells or cells in early development and is induced during multicellular development with kinetics similar to those of SP60 gene expression. This activity is also induced in response to cAMP and specifically binds the wild-type CAE-1- and CAE-2-containing oligonucleotides. CAE-1 and CAE-2 oligonucleotides containing point mutations within the CAE core sequence neither bind to nor compete for the cAMP-induced, developmentally regulated factor(s) and result in substantial reductions in expression levels when substituted for the wild-type CAEs in vivo. The correlation between in vitro binding and in vivo function suggests that the CAE-1/CAE-2 binding activity may be involved in regulating cAMP and developmentally induced expression of SP60. A second, constitutive in vitro binding activity with high affinity to CAE-3 is also described. Models are proposed to relate the binding activities with the effects of the mutations on the spatial patterning of SP60-lacZ expression.
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PMID:Developmental and spatial regulation of a Dictyostelium prespore gene: cis-acting elements and a cAMP-induced, developmentally regulated DNA binding activity. 166 78

Fragments of the rat ferritin-H 5'-flanking region up to 1 kilobase in length were generated by the polymerase chain reaction using FRTL5 rat thyroid cell genomic DNA as template. Ferritin-H 5'-flanking region fragments of 219, 351, 666, and 1046 basepairs (bp), ligated up-stream to the reporter gene luciferase, were transiently transfected into FRTL5 thyroid cells and NIH-3T3 mouse fibroblasts. In both cell types, constitutive (nonstimulated) ferritin-H promoter activity increased progressively with constructs containing increasing lengths of 5'-flanking region. TSH or (Bu)2cAMP (dBcAMP) stimulation of FRTL5 cells transfected with the shorter (219 and 351 bp) ferritin-H 5'-flanking region fragments increased promoter activity 2- to 3-fold. However, with the longer DNA segments (666 and 1046 bp), the extent of TSH stimulation was less. Exposure of transfected NIH-3T3 cells to dBcAMP mimicked in all respects the effects of TSH and dBcAMP on ferritin-H promoter activity in FRTL5 cells. Transcription initiation sites in the luciferase reporter gene were unaffected by the length of the ferritin-H 5'-flanking region included in the construct or by dBcAMP stimulation. Plasmid constructs with 45 bp of the ferritin-H 5'-flanking region containing a potential cAMP response element did not reveal any promoter activity or dBcAMP responsiveness in this region. Gel shift mobility assays with the -219 bp ferritin-H 5'-flanking region fragment and NIH-3T3 nuclear proteins revealed specific protein-DNA interaction. Reduced DNA mobility was inhibited by excess unlabeled probe DNA, but not by DNA fragments corresponding to the recognition sites for a variety of known trans-activating factors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thyrotropin and adenosine 3',5'-monophosphate stimulate the activity of the ferritin-H promoter. 196 70

The gene encoding angiotensinogen, the glycoprotein precursor of the potent vasopressor peptide angiotensin II, is transcriptionally activated in hepatocytes during the acute-phase response through interactions of mutually cooperative glucocorticoid receptors and proteins that bind to an acute-phase response element (APRE) 5'-AGTTGGGATTTCCCAACC-3'. The APRE binds a family of constitutive proteins (BPcs) and a cytokine inducible protein (BPi) that is indistinguishable from nuclear factor kappa B (NF kappa B). The interactions of purified proteins with the APRE were studied by in vitro binding and in vivo transcriptional trans-activation assays. BPc is a family of heat-stable DNA binding proteins, the different sized members of which are capable of forming heterodimers. BPcs are recognized by anti-C/EBP antiserum and produce a footprint similar to bacterially expressed C/EBP on the APRE. BPi has a 4- to 5-fold greater affinity for the APRE than the BPcs, and contacts guanosine residues distinct from those contacted by the BPcs, demonstrating that these two classes of proteins contain functionally distinct DNA binding domains. Assays of APRE-luciferase reporter plasmids transfected into HepG2 cells show that a mutated APRE that binds only BPi functions as an IL-1 alpha inducible enhancer, whereas a mutated APRE that binds only BPc does not. The APRE mutant that binds the C/EBP-like BPcs to the exclusion of BPi functions as an uninducible basal enhancer both in the native context of the angiotensinogen gene and when multimerized and placed upstream of a minimal angiotensinogen promoter. The wild-type APRE that binds both BPi and BPc is less inducible by IL-1 alpha than the mutated APRE that binds only BPi. Gel shift competition assays demonstrate in vitro that the mechanism of transcriptional regulation by the APRE involves a competition between BPc and the inducible BPi for binding to the APRE. IL-1 alpha stimulation of hepatocytes leads to nuclear translocation of the NF kappa B-like BPi which competes with the constitutive C/EBP-like BPcs for overlapping binding sites on the APRE and thereby replaces weak transcriptional activators with a stronger one.
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PMID:A family of constitutive C/EBP-like DNA binding proteins attenuate the IL-1 alpha induced, NF kappa B mediated trans-activation of the angiotensinogen gene acute-phase response element. 217 52

A system for inducible gene expression by vaccinia virus (VV) vectors utilizing the Escherichia coli lac I repressor/operator system is described. A VV recombinant that expresses the lac I repressor protein from the constitutively active 7.5K promoter was constructed. Gel retardation experiments showed that a protein present in extracts of cells infected with this virus, but not wild-type virus, bound to the lac operator and that this binding was inhibited by IPTG. A series of VV recombinants were constructed that contained a 21-bp synthetic operator sequence(s) at different positions between the VV late 4b promoter and the firefly luciferase gene. Cells were co-infected with one of these viruses and the VV recombinant expressing the lac I repressor protein in the presence or absence of IPTG, and the level of luciferase activity was determined. Single operators positioned 19, 11, or 6 bp downstream of the promoter resulted in the 30, 50, or 97% inhibition of luciferase activity, respectively, while two operators increased the inhibition to greater than 99.9%. Addition of 1.25 mM IPTG at any time after infection restored 90% of enzyme activity from viruses containing a single operator, but reversal was only 50% when two operators were present. Both elements of the lac I inducible system were functional and stable in the genome of single recombinant virus. S1 nuclease protection of virus mRNA confirmed that luciferase expression was controlled at the transcriptional level and that IPTG did not affect transcription of endogenous VV genes. The utility of this inducible expression system for functional analyses of endogenous VV genes is demonstrated by the controlled expression of a gene encoding a 14-kDa protein and the correlation of 14-kDa expression with a biological property of the virus, namely plaque size phenotype. Plasmid vectors that are generally applicable to the inducible expression of genes by VV recombinants are described.
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PMID:Inducible gene expression from vaccinia virus vectors. 219 97

Insulin-like growth factor-binding protein-1 (IGFBP-1) is produced by the liver and regulated by glucocorticoids and insulin at the level of gene transcription. To identify DNA sequences mediating the effects of glucocorticoids and insulin on IGFBP-1 promoter activity we created luciferase reporter gene constructs and performed transfection studies in H4IIE hepatoma cells. Initial studies confirmed that the IGFBP-1 promoter is functional when inserted in the correct orientation, but not in the reverse orientation. Dexamethasone (DEX) increased promoter activity 10-fold, and insulin reversed this effect of DEX by 85% at 8 h. The effects of DEX were abolished when constructs were truncated to 89 bases from the RNA cap site, and DNase footprinting with the DNA-binding domain of the human glucocorticoid receptor identified an imperfect palindrome containing two receptor-binding sites separated by three nucleotides typical of a glucocorticoid response element (GRE) at this location. Mutation of either binding site (or half-site) disrupted the effects of DEX, confirming that this sequence functions as a GRE. Two other regions of the promoter also footprinted with the glucocorticoid receptor protein and contained sequences consistent with glucocorticoid receptor-binding sites; however, neither of these footprints contained the full structure expected of a functional GRE, and neither mutation nor deletion of these other sequences altered the effects of DEX on promoter activity. To identify the DNA sequences required for the effects of insulin on glucocorticoid-stimulated promoter activity, we created internal deletions throughout the IGFBP-1 promoter region. Deletion of the 22-basepair (bp) sequence immediately 5' from the GRE disrupted the effect of insulin and appeared to increase basal promoter activity at least 2-fold in each of eight experiments (P < 0.001 vs. intact promoter). This region of the IGFBP-1 promoter contains a 19-bp palindrome (CAAAACAAACTTATTTTG) that overlaps the 5'-end of the GRE and is fully conserved in the human IGFBP-1 promoter. Each half of this palindrome resembles previously identified insulin response sequences, and deletion/mutation analysis suggests that each half may contribute to the effects of insulin on promoter activity. Gel shift studies confirmed that this palindrome binds H4IIE nuclear proteins. In summary, we have identified a GRE in the 5'-promoter region of the rat IGFBP-1 gene approximately 90 bp up-stream from the RNA cap site as well as a contiguous 22-bp region that plays a critical role in mediating the effects of insulin on glucocorticoid-stimulated promoter activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter. 750 35

To define the minimal promoter responsible for expression of CD18 in myeloid and lymphoid cells, we generated 5' and 3' deletion constructs of a segment extending 785 bp upstream and 19 bp downstream of a major transcription start site and determined their effects on driving expression of the luciferase reporter gene in transfected hematopoietic cell lines. A region extending from nucleotides (nt) -302 to +19 was sufficient for cell-restricted and phorbol ester-inducible expression. DNase I footprinting of this region revealed two adjacent protected segments extending from nt -81 to -68 (box A) and -55 to -41 (box B). When a construct of 47 nt in length containing box A and box B and lacking other 3' or 5' elements was cloned into a promoterless vector, it conferred tissue-specific and phorbol ester-inducible expression. Gel retardation revealed that the protein components of two major protein-DNA complexes that form on both box A and box B and are required for transcriptional activation are members of the Ets oncoprotein family; one is related to the GA-binding protein (GABP), and the other is related to PU.1/Spi-1. The minimal CD18 promoter, lacking TATA, CAAT, and initiator elements and consisting primarily of Ets repeats, may exemplify an emerging class of promoters with which the concerted binding of Ets factors is necessary and sufficient to mediate transcriptional activation through direct recruitment of the basal transcription machinery.
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PMID:The human beta 2 integrin CD18 promoter consists of two inverted Ets cis elements. 751 Dec 9

Cell line-dependent expression of foreign genes in the baculovirus system was investigated using a recombinant vAc beta hCG-luc virus carrying two reporter genes--beta subunit of human chorionic gonadotropin (beta hCG) and luciferase (luc)--placed under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoters. Five different lepidopteran cell lines derived from Spodoptera frugiperda (Sf21 and Sf9), Bombyx mori (BmN and Bm5), and Trichoplusia ni (TN368) were used as host cells. TN368 expressed both beta hCG and LUC to maximum levels, followed by BmN, Sf21, and Sf9 in descending order. Bm5 did not show any evidence of synthesis of the two proteins. Dot blot analysis of DNA from the vAc beta hCG-luc-infected cells revealed that the level of entry of viral DNA was the same for all the five cell lines. After the completion of viral DNA replication (18 hr post infection), the level of viral DNA was the same for all the cell lines except for Bm5 where viral DNA replication did not take place and the residual virus was cleared from the cells. Analysis of RNA from the four expressing cell lines revealed a direct correlation between protein levels and levels of mRNA, suggesting transcriptional control. Differences in mRNA stability between cell lines was also evident. Gel retardation analysis of a host factor binding to transcriptionally important sequence motifs within the AcNPV polyhedrin gene promoter revealed an inverse correlation between the levels of this polyhedrin promoter-binding protein (PPBP) and reporter gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transcriptional regulation of cell line-dependent, baculovirus-mediated expression of foreign genes. 753 Apr 52


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