Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple and economical large-scale in vivo screen for firefly luciferase expression in transgenic zebrafish is described. The screen is a film assay of luminescence during embryogenesis. Either luciferin substrate can be microinjected into the embryo, or the embryo can be raised in a luciferin solution. In a test of transient expression in the G0 (microinjected) generation, a construct with the human cytomegalovirus (CMV) promoter gave higher levels of expression than three other constructs. Using the CMV promoter, injection of supercoiled or linear DNA led to approximately equivalent amounts of expression. Although G0 transient luciferase expression is high enough to be reliably screened, G1 integrated expression is either low or nonexistent, and therefore unscreenable. In the G1 and G2 generations, low-level expression was increased with application of 5-azacytidine. The fact that both transgene methylation and 5-azacytidine activation of expression occurred suggests that methylation is involved in either reducing or eliminating integrated luciferase expression. This in vivo luciferase screen may be useful for insertional mutagenesis, promoter, gene, or enhancer traps, promoter analysis, and optimization of conditions for gene transfer.
Mol Mar Biol Biotechnol 1994 Dec
PMID:An in vivo screen for the luciferase transgene in zebrafish. 753 26

Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug-sensitive and drug-resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.
Mol Microbiol 1995 Mar
PMID:L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria. 762 62

Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.
Mol Cell Biol 1995 Sep
PMID:Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia. 765 5

Retinoids are reported to stimulate apolipoprotein (apo) A-I gene promoter activity (Rottman et al. 1991. Mol. Cell. Biol. 11: 3814-3820) and apoA-I protein secretion by monkey hepatocytes (Kaptein et al. 1993. Arterioscler. Thromb. 13: 1505-1514). In this study we have assessed the effects of retinoids on parameters of apoA-I biosynthesis in human cell lines. Caco-2 and HepG2 cells (human intestinal and hepatoma cell lines, respectively, both known to express and secrete apoA-I) were stably transfected with a reporter gene construct containing 1.3 kb of the 5-'flanking region of the human apoA-I gene linked to the firefly luciferase coding region. These cells were incubated for 48 h with 10 microM all-trans retinoic acid (RA) or 9-cis RA. The cells were then assayed for luciferase activity, for apoA-I mRNA level, and for secretion of apoA-I protein in the medium. Secretion of apoB was monitored as well. In Caco-2 cells, all-trans and 9-cis RA increased luciferase activity, mRNA content, and protein secretion by 40% to 80% above control. Strikingly, in HepG2 cells all-trans and 9-cis RA caused a more marked stimulation of luciferase activity (by 100-150%) but a weaker increase of mRNA content and protein secretion (by 25-30%). In contrast, apoB secretion was inhibited by the two retinoids in Caco-2 cells and not changed in HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of human apolipoprotein A-I expression in Caco-2 and HepG2 cells by all-trans and 9-cis retinoic acids. 765 49

The cytotoxic efficacy of antitumor drugs targeted at DNA topoisomerase II (topo II) in many cases varies in direct proportion to cellular topo II content. To investigate the transcriptional control of the predominant alpha form of topo II, the 5' flanking region of the human topo II alpha gene (positions -562 to +90) was subcloned into a firefly luciferase reporter plasmid and transiently transfected into HL-60 human leukemia cells, a line capable of monocytic differentiation after treatment with various agents. Early in phorbol-12-myristate-13-acetate (30 nM)-induced differentiation (18-24 hr after treatment), an unexpected 3-5-fold activation of topo II alpha gene promoter activity was observed. Activation was observed in HL-60 cells and U-937 cells, but not in HeLa human cervical carcinoma cells. Sodium butyrate (NaB) (0.4 mM) also led to activation (4-17-fold) of the topo II alpha promoter in HL-60 and U-937 cells. Promoter sequences between position -90 and position +90 mediated the inducing effects of NaB. This NaB-dependent promoter-reporter induction was partly mirrored by a transient approximately 2-fold increase in endogenous topo II alpha enzyme. The stimulus for promoter activation could be partly attributed to a 2-fold increase in DNA synthesis at 16 hr for NaB, but not phorbol-12-myristate-13-acetate. Regardless of the primary stimulus for topo II alpha promoter trans-activation, it could be bypassed by treatment of HL-60 cells with NaB for 48 hr before transfection, revealing the expected 60-70% suppression of topo II alpha promoter activity. Further study of topo II alpha promoter down-regulation later in monocytic differentiation may serve as a model for elucidating the transcriptional mechanisms that may also be exploited by tumor cells expressing intrinsic or acquired resistance to topo II-directed drugs.
Mol Pharmacol 1995 Apr
PMID:Topoisomerase II alpha promoter trans-activation early in monocytic differentiation of HL-60 human leukemia cells. 772 30

Thyroid hormone (T3) stimulates Na,K-ATPase activity and alpha and beta subunit mRNA abundances in myocardial cells in vivo and in vitro. In this study, we used transient transfection and nuclear run-on assays to determine whether T3 regulates the transcription rate of the Na,K-ATPase alpha 2 subunit gene. Primary cultures of neonatal rat cardiac myocytes were incubated with 100 nM T3 for 1, 3, and 6 d, and alpha 2 mRNA levels were measured by Northern blot hybridization analysis. There was no change in the abundance of alpha 2 mRNA by 1 d of T3 treatment, whereas a two- and threefold increase in alpha 2 mRNA was evident when cells were exposed to T3 for 3 and 6 d, respectively. A portion of the rat alpha 2 gene containing 1700 base pairs (bp) of 5'-flanking DNA sequence was isolated and fused to the firefly luciferase gene. Transient transfection experiments utilizing this chimeric gene showed no T3 trans-activation of reporter gene activity either in the absence or presence of cotransfected beta 1 or alpha 1 isoforms of rat T3 receptor (T3R). In contrast, cotransfection of T3R facilitated a strong stimulation of luciferase activity driven by a construct containing a single copy of a palindromic T3 response element (TRE). Nuclear run-on analysis indicated that the rate of transcription of the endogenous alpha 2 gene was enhanced 1.2-fold at 3 d of T3 treatment, and was not regulated at either 1 or 6 d. These results indicate that the T3-dependent increase in alpha 2 mRNA content at 6 d is mediated at a post-transcriptional level. Unexpectedly, we observed a T3-dependent three-to sixfold repression of alpha 2/luciferase expression in cardiac myocytes cotransfected with T3R. Deletion analysis of the 5' end of the alpha 2 gene revealed a negative TRE between nucleotides -354 and -100.
Cell Mol Biol Res 1994
PMID:Thyroid hormone regulation of Na,K-ATPase alpha 2 gene expression in cardiac myocytes. 780 25

We investigated the onset of paternal gene expression in the early mouse embryo. We obtained transgenic mouse embryos by fertilizing BD (C57BL/6N x DBA) F1 hybrid female oocytes in vitro, with sperm from homozygous transgenic males carrying integrated chicken beta-actin promoter-driven firefly luciferase cDNA. We then examined the RNA and protein synthesis of the luciferase gene in embryos from the 1- to 2-cell stage. RNA transcripts of the luciferase gene were first detected in the 1-cell stage embryos as early as 13 hr postinsemination, just prior to elongation. By photon-count imaging, functional luciferase was identified at the 2-cell stage 23 hr postinsemination. These findings indicate that the paternal endogenous gene is already transcribed in the late 1-cell embryos, although paternally derived protein is not synthesized until the 2-cell stage. Therefore, these results suggest that the embryonic gene is activated as early as the late 1-cell stage.
Mol Reprod Dev 1994 Oct
PMID:Onset of paternal gene activation in early mouse embryos fertilized with transgenic mouse sperm. 782 13

Candida albicans WO-1 switches spontaneously, frequently, and reversibly between a hemispherical white and a flat gray (opaque) colony-forming phenotype. This transition affects a number of morphological and physiological parameters and involves the activation and deactivation of phase-specific genes. The WH11 gene is transcribed in the white but not the opaque phase. A chimeric WH11-firefly luciferase gene containing the 5' upstream region of WH11 was demonstrated to be under phase regulation regardless of the site of integration, and a series of promoter deletion constructs was used to delineate two white-phase-specific transcription activation domains. Gel retardation experiments with the individual distal or proximal domain and white-phase or opaque-phase protein extract demonstrated the formation of one distal white-phase-specific complex and two proximal white-phase-specific complexes. Specific subfragments were tested for their ability to compete with the entire domain in the formation of complexes with white-phase protein extract in order to map the proximal domain sequence involved in white-phase-specific complex formation. Our results indicate that white-phase-specific transcription of WH11 is positively regulated by trans-acting factors interacting with two cis-acting activation sequences in the WH11 promoter.
Mol Cell Biol 1995 Mar
PMID:Functional analysis of the promoter of the phase-specific WH11 gene of Candida albicans. 786 69

Tyrosinase is a rate-limiting enzyme in melanin biosynthesis and is specifically expressed in differentiated melanocytes. We have identified the enhancer element in the 5'-flanking region of the human tyrosinase gene that is responsible for its pigment cell-specific transcription and have termed it tyrosinase distal element (TDE) (positions -1861 to -1842). Transient expression assays showed that TDE confers efficient expression of a firefly luciferase reporter gene linked to the tyrosinase gene promoter in MeWo pigmented melanoma cells but not in HeLa cells, which do not express tyrosinase. TDE was specifically bound by nuclear proteins of MeWo and HeLa cells, the binding properties of which were indistinguishable in gel mobility shift assays. TDE contains the CATGTG motif in its center, and mutation analysis indicates that the CA dinucleotides of this motif are crucial for protein binding and pigment cell-specific enhancer function. The CATGTG motif is consistent with the consensus sequence recognized by a large family of transcription factors with a basic helix-loop-helix structure, which prompted us to examine the possible involvement of a ubiquitous transcription factor, USF, and a novel factor, microphthalmia-associated transcription factor (MITF), recently cloned as the human homolog of the mouse microphthalmia (mi) gene product. The mi phenotype is associated with a mutant mi locus and characterized by small eyes and loss of melanin pigments. Both USF and MITF are predicted to contain a basic helix-loop-helix structure and a leucine zipper structure. We provide evidence that USF binds to TDE, whereas we were unable to detect the DNA-binding activity of MITF. Transient coexpression assays showed that MITF specifically transactivates the promoter activity of the tyrosinase gene through the CATGTG motif of TDE but not the promoter of the ubiquitously expressed heme oxygenase gene, while USF is able to activate both promoters. These results indicate that MITF is a cell-type-specific factor that is capable of activating transcription of the tyrosinase gene.
Mol Cell Biol 1994 Dec
PMID:Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene. 786 73

Efficient antimineralocorticoid selection requires a reliable, discriminating and easy assay for monitoring biological activity of not only the specific receptor, but also closely related receptors such as glucocorticoid and progestin. These related activities should be as low as possible to obtain specific antimineralocorticoid compounds. In this paper, we describe two cellular models used for easy and specific measurement of mineralocorticoid and progestin activities. These models involve the induction of firefly luciferase under hormonal control mediated by a chimeric receptor. The first model comprises transiently transfected MCF-7 cells, whereas the second uses stably transfected HeLa cells. Glucocorticoid activity was assayed with the classic tyrosine-aminotransferase induction method in HTC cells. Six compounds of a new family of 11 beta-substituted-17-spirolactone steroids were thus studied and compared to control compounds. Five of them showed antimineralocorticoid activity and one was active at a concentration lower than that of mespirenone.
J Steroid Biochem Mol Biol 1994 May
PMID:Development of specific bioluminescent in vitro assays for selecting potential antimineralocorticoids. 800 37


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