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Luciferase Gene (luciferase + gene)
Selected AbstractsIdentification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv. trifoliiFEBS JOURNAL, Issue 24 2000Hwan Young Lee A novel gene, matR, located upstream of matABC, transcribed in the opposite direction, and encoding a putative regulatory protein by sequence analysis was discovered from Rhizobium leguminosarum bv. trifolii. The matA, matB, and matC genes encode malonyl-CoA decarboxylase, malonyl-CoA synthetase, and a presumed malonate transporter, respectively. Together, these enzymes catalyze the uptake and conversion of malonate to acetyl-CoA. The deduced amino-acid sequence of matR showed sequence similarity with GntR from Bacillus subtilis in the N-terminal region encoding a helix-turn-helix domain. Electrophoretic mobility shift assay indicated that MatR bound to a fragment of DNA corresponding to the mat promoter region. The addition of malonate or methylmalonate increased the association of MatR and DNA fragment. DNase I footprinting assays identified a MatR binding site encompassing 66 nucleotides near the mat promoter. The mat operator region included an inverted repeat (TCTTGTA/TACACGA) centered ,46.5 relative to the transcription start site. Transcriptional assays, using the luciferase gene, revealed that MatR represses transcription from the mat promoter and malonate alleviates MatR-mediated repression effect on the expression of Pmat -luc+ reporter fusion. [source] Direct DNA delivery into zebrafish embryos employing tissue culture techniquesGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2001Raquel Sussman Abstract Summary: The production of transfected fish embryos requires expertise in injecting the fertilized eggs and/or expensive equipment for electroporation or microprojectiles. This article demonstrates that by exposure to DNA constructs conjugated with transfecting reagents dechorionated Danio rerio embryos are capable of acquiring extracellular DNA and expressing reporter genes. Embryos incubated with pCMVluc complexed with GeneJammer or GenePORTER expressed luciferase 24,48 h after exposure. pCMVGFP DNA mixed with the same agents generated embryos that exhibited differential patterns of expression of green fluorescent protein (GFP). Embryonic development varied depending on the procedure employed and the reporter gene utilized. Expression of the luciferase gene did not interfere with the subsequent development of the embryos. In contrast, the embryos expressing a high level of GFP were affected, probably due to a very active promoter. These results demonstrate the ease of obtaining transfected fish embryos, which facilitate the mass production of new genotypes and extend the procedure to laboratories with limited resources. genesis 31:1,5, 2001. © 2001 Wiley-Liss, Inc. [source] Severe pulmonary metastasis in obese and diabetic miceINTERNATIONAL JOURNAL OF CANCER, Issue 12 2006Akinori Mori Abstract Although obesity is known as a risk factor for several human cancers, the association of obesity with cancer recurrence and metastasis remains to be characterized. Here, B16-BL6 melanoma and Lewis lung carcinoma cells were intravenously injected into diabetic (db/db) and obese (ob/ob) mice. The number of experimental lung colonies was markedly promoted in these mice when compared with C57BL/6 mice. In contrast, tumor growth at the implanted site was comparable when cells were inoculated orthotopically. The use of B16-BL6 cells stably transfected with the luciferase gene revealed that the increased metastasis reflected a difference mainly within 6 hr after the intravenous inoculation of tumor cells. Administration of recombinant leptin in ob/ob mice abolished the increase in metastasis early on as well as the decrease in the splenic NK cell number. In addition, depletion of NK cells by an anti-asialo-GM1 antibody abrogated the enhanced metastasis in db/db mice. These results demonstrate that metastasis is markedly promoted in diabetic and obese mice mainly because of decreased NK cell function during the early phase of metastasis. © 2006 Wiley-Liss, Inc. [source] Genkwanin up-regulates the transcriptional activation of human type vii collagen gene promoterINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 4 2007N. Takebayashi In a recent study, stimulating the formation of anchoring fibrils at the basement membrane zone in skin contributed to preventing skin ageing, such as wrinkle formation. Expression of the type VII collagen gene induces the formation of anchoring fibrils composed mainly of collagen type VII. We therefore transiently transfected a keratinocyte cell line with the plasmids containing type VII collagen gene promoter located upstream of the luciferase gene. We investigated the promoter activity under the presence of flavonoids and we found that Genkwanin up-regulates the transcriptional activation of human type VII collagen gene promoter. [source] Hepatocyte Growth Factor Contributes to Fracture Repair by Upregulating the Expression of BMP Receptors,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2005Yuuki Imai MD Abstract Hepatocyte growth factor (HGF) is activated and the expression of BMP receptors (BMPRs) is induced around the fracture site during the early phase of fracture repair. HGF facilitates the expression of BMPRs in mesenchymal cells. This study suggests that HGF contributes to fracture repair by inducing the expression of BMPRs. Introduction: The precise mechanisms that control the upregulation of BMP, BMPRs, and other molecules involved in bone repair are not completely understood. In this study, we hypothesized that HGF, activated through the action of thrombin on the HGF activator, may enhance BMP action through the local induction of BMP or BMPRs. Materials and Methods: Callus samples from tibial fractures in mice were harvested for immunohistochemical analysis of HGF and phosphorylated c-Met, for in situ hybridization of BMPRs, and for real-time RT-PCR analysis for the expression of HGF, c-Met, and BMPRs. To study the changes in gene expression of BMPRs in response to HGF, C3H10T1/2 cells were cultured with or without HGF and harvested for real-time RT-PCR and for Western blot analysis. To evaluate the contribution of HGF to the biological action of BMP2, C3H10T1/2 cells and primary muscle-derived mesenchymal cells were precultured with HGF and cultured with BMP2. In addition, the expression of the luciferase gene linked to the Id1 promoter containing the BMP responsive element and alkaline phosphatase (ALP) activity were assayed. Results: Positive immunostaining of HGF and phosphorylated c-Met was detected around the fracture site at 1 day after the fracture was made. mRNA expression of BMPRs was increased 1 day after fracture and localized in mesenchymal cells at the fracture site. From an in vitro study, the expression of mRNA for BMPRs was elevated by treatment with HGF, but the expression of BMP4 did not change. Western blot analysis also showed the upregulation of BMPR2 by HGF treatment. The results from the luciferase and ALP assays indicated increased responsiveness to BMPs by treating with HGF. Conclusions: This study indicates that HGF is activated and expressed at the fracture site and that HGF induces the upregulation of BMPRs in mesenchymal cells. Furthermore, HGF may facilitate BMP signaling without altering the expression of BMP molecules. [source] Cloning and molecular dissection of the 8.8 kb pig uroplakin II promoter using transgenic mice and RT4 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Deug-Nam Kwon Abstract Uroplakin II (UPII) gene expression is highly tissue and cell specific, with mRNA present in the suprabasal cell layers of the bladder and urethra. Previous reports described the mouse UPII (mUPII) promoter as primarily urothelium selective. However, ectopic expression of a transgene under the 3.6 kb mUPII promoter was also detected in brain, kidney, and testis in some transgenic mouse lines. Here, we have cloned an 8.8 kb pig UPII (pUPII) promoter region and investigated which cells within the bladder and urethra express a transgene consisting of the pUPII promoter fused to human erythropoietin (hEPO) or a luciferase gene. pUPII-luciferase expression vectors with various deletions of the promoter region were introduced into mouse fibroblast (NIH3T3), Chinese hamster ovary (CHO), and human bladder transitional carcinoma (RT4). A 2.1 kb pUPII promoter fragment displayed high levels of luciferase activity in transiently transfected RT4 cells, whereas the 8.8 kb pUPII promoter region displayed only low levels of activity. The pUPII-hEPO expression vector was injected into the pronucleus of zygotes to make transgenic mice. To elucidate the in vivo molecular mechanisms controlling the tissue- and cell-specific expression of the pUPII promoter gene, transgenic mice containing 2.1 and 8.8 kb pUPII promoter fragments linked to the genomic hEPO gene were generated. An erythropoietin (EPO) assay showed that all nine transgenic lines carrying the 8.8 kb construct expressed recombinant human erythropoietin (rhEPO) only in their urethra and bladder, whereas two transgenic lines carrying the 2.1 kb pUPII promoter displayed hEPO expression in several organs including bladder, kidney, spleen, heart, and brain. These studies demonstrate that the 2.1 kb promoter contains the DNA elements necessary for high levels of expression, but lacks critical sequences necessary for tissue-specific expression. We compared binding sites in the 2.1 and 8.8 kb promoter sequences and found five peroxisome proliferator responsive elements (PPREs) in the 8.8 kb promoter. Our data demonstrated that proliferator-activated receptor (PPAR)-, activator treatment in RT4 cells induced the elevated expression of hEPO mRNA under the control of the 8.8 kb pUPII promoter, but not the 2.1 kb promoter. Collectively, our data suggested that all the major trans-regulatory elements required for bladder- and urethra-specific transcription are located in the 8.8 kb upstream region and that it may enhance tissue-specific protein production and be of interest to clinicians who are searching for therapeutic modalities with high efficacy and low toxicity. J. Cell. Biochem. 99: 462,477, 2006. © 2006 Wiley-Liss, Inc. [source] HER2 signaling enhances 5,UTR-mediated translation of c-Myc mRNAJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004Enrico Galmozzi The increased levels of c-Myc protein observed previously in an ovarian carcinoma cell line stably transfected to express HER2 has suggested a role for the HER2 pathway in c-Myc expression. Analysis of HER2-transfected cells stimulated with heregulin ,1 (HRG) revealed increased c-Myc protein levels but not a corresponding increase in c-Myc mRNA expression or any change in c-Myc protein half-life. Transfection of HER2-overexpressing cells with a construct containing the 5, untranslated region (5,UTR) of c-Myc mRNA originated from the P2 promoter and placed upstream of the Renilla luciferase gene, enhanced reporter expression upon stimulation with HRG. The HRG-mediated increase in reporter activity correlated with the HRG-mediated induction observed for c-Myc protein, identifying the P2-derived leader (P2L) of c-Myc mRNA as the cis -element involved in c-Myc translational induction. Both the increase in c-Myc protein levels and P2L-enhanced translational activity were inhibited by the PI3K inhibitor wortmannin. Together, these results demonstrate that HRG stimulation of HER2 overexpressing cells leads to enhanced c-Myc protein synthesis through activation of the PI3K/Akt/mTOR pathway and that the P2L of c-Myc mRNA is the element responsible for induction of c-Myc translation. © 2004 Wiley-Liss, Inc. [source] In vitro analysis of synergism and antagonism of different nucleoside/nucleotide analogue combinations on the inhibition of human immunodeficiency virus type 1 replication,JOURNAL OF MEDICAL VIROLOGY, Issue 2 2009M. Perez-Olmeda Abstract In this study we have developed an in vitro system to evaluate the combined effect of two NRTIs on HIV replication and to assess their antagonism or synergy. Synergy or antagonism effect was determined in peripheral blood mononuclear cells (PBMCs) to approach a more physiological model than T-cell lines. PBMCs were infected with a full-length HIV-1 clone carrying the luciferase gene as a reporter. The following combinations were investigated: zidovudine+stavudine (ZDV,+, d4T), lamivudine,+,abacavir (3TC,+,ABC), lamivudine,+,didanosine (3TC,+,ddI), lamivudine,+ stavudine (3TC,+,d4T), tenofovir,+,stavudine (TDF,+,d4T), tenofovir,+,didanosine (TDF,+,ddI), tenofovir,+,abacavir (TDF,+,ABC), tenofovir,+, lamivudine (TDF,+,3TC), tenofovir,+,zidovudine (TDF,+,ZDV), stavudine,+,didanosine (d4T,+,ddI), zidovudine,+,lamivudine (ZDV,+,3TC), abacavir,+, didanosine (ABC,+,ddI), zidovudine,+,didanosine (ZDV,+,ddI), and abacavir,+,stavudine (ABC,+, d4T). The effect of combining two drugs was evaluated with a quantitative method based on the median-effect principle of Chou and Talalay. A synergistic effect was observed with combinations containing TDF and ZDV or d4T, d4T and ddI and ZDV plus 3TC. In contrast, combinations including TDF,+,ddI, 3TC,+,ddI, ABC,+,ddI, and ZDV,+,ddI showed an antagonistic effect on the inhibition of viral replication at all levels of inhibition tested. Lower antagonistic effect was also found in drug combinations that included 3TC,+,ABC, 3TC,+,TDF, 3TC,+,d4T, and TDF,+, ABC. In conclusion, the method developed allows to measure in vitro the effect of different combinations of two NRTIs on HIV replication. The results suggest that combined therapy including TDF with thymidine analogues may be considered for future therapeutic options in contrast to clearly antagonistic combinations such us TDF plus ddI or 3TC plus ddI, that would explain virological failure in clinical studies when these combinations were used. J. Med. Virol. 81:211,216, 2009. © 2008 Wiley-Liss, Inc. [source] The effect of a promoter polymorphism on the transcription of nitric oxide synthase 1 and its relevance to Parkinson's diseaseJOURNAL OF NEUROSCIENCE RESEARCH, Issue 10 2009Terrie Rife Abstract Transcriptional changes of the enzyme nitric oxide synthase I (NOS1) are believed to play a role in the development of many diseases. The gene for NOS1 has 12 alternative first exons (1A,1L). The 1F exon is one of the most highly utilized first exons in the brain and has a polymorphism ((TG)mTA(TG)n) located in its promoter region. The polymorphism's length has been suggested to affect NOS1 transcription and play a role in Parkinson's disease (PD); however, the actual influence of the polymorphism on NOS1 transcription has not been studied. To better characterize the links of the polymorphism with PD, a genotyping study was done comparing polymorphism length among 170 PD patients and 150 age-matched controls. The pattern of changes between the two group's allele frequencies shows statistical significance (P = 0.0359). The smallest polymorphism sizes are more predominant among PD patients than controls. To study the effects of this polymorphism on NOS1 gene transcription, reporter gene constructs were made by cloning the NOS1 1F promoter with polymorphism lengths of either 42, 54, or 62 bp in front of the luciferase gene and transfecting them into HeLa or Sk-N-MC cells. NOS1-directed reporter gene constructs with the 62-bp polymorphism increased transcription of luciferase 2.2-fold in HeLa and 1.8-fold in Sk-N-MC cells compared with reporter gene constructs with the 42-bp polymorphism. These data suggest that if smaller polymorphism size contributes to the higher NOS1 levels in PD patients, an as yet unknown transcriptional mechanism is required. © 2009 Wiley-Liss, Inc. [source] In vivo bioluminescence imaging study to monitor ectopic bone formation by luciferase gene marked mesenchymal stem cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 7 2008Cristina Olivo Abstract Mesenchymal stem cells (MSCs) represent a powerful tool for applications in regenerative medicine. In this study, we used in vivo bioluminescence imaging to noninvasively investigate the fate and the contribution to bone formation of adult MSCs in tissue engineered constructs. Goat MSCs expressing GFP-luciferase were seeded on ceramic scaffolds and implanted subcutaneously in immune-deficient mice. The constructs were monitored weekly with bioluminescence imaging and were retrieved after 7 weeks to quantify bone formation by histomorphometry. With increasing amounts of seeded MSCs (from 0 to 1,×,106 MSC/scaffold), a cell-dose related increase in bioluminescence was observed at all time points, correlating with increased bone formation at 7 weeks. To investigate the relevance of MSC proliferation to bone deposition, cell-seeded scaffolds were irradiated. The irradiated cells were functional with respect to oxygen consumption but no increase in bioluminescence was observed in vivo, and only minimal bone was produced. Proliferating MSCs are likely required for initiation of bone formation in tissue engineered constructs in vivo. Bioluminescence is a useful tool to monitor cellular responses and predict bone formation in vivo. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:901,909, 2008 [source] OLIGONUCLEOTIDE PRIMERS FOR THE DETECTION OF BIOLUMINESCENT DINOFLAGELLATES REVEAL NOVEL LUCIFERASE SEQUENCES AND INFORMATION ON THE MOLECULAR EVOLUTION OF THIS GENE,JOURNAL OF PHYCOLOGY, Issue 2 2008Andrea Baker Bioluminescence is reported in members of 18 dinoflagellate genera. Species of dinoflagellates are known to have different bioluminescent signatures, making it difficult to assess the presence of particular species in the water column using optical tools, particularly when bioluminescent populations are in nonbloom conditions. A "universal" oligonucleotide primer set, along with species and genus-specific primers specific to the luciferase gene were developed for the detection of bioluminescent dinoflagellates. These primers amplified luciferase sequences from bioluminescent dinoflagellate cultures and from environmental samples containing bioluminescent dinoflagellate populations. Novel luciferase sequences were obtained for strains of Alexandrium cf. catenella (Whedon et Kof.) Balech and Alexandrium fundyense Balech, and also from a strain of Gonyaulax spinifera (Clap. et Whitting) Diesing, which produces bioluminescence undetectable to the naked eye. The phylogeny of partial luciferase sequences revealed five significant clades of the dinoflagellate luciferase gene, suggesting divergence among some species and providing clues on their molecular evolution. We propose that the primers developed in this study will allow further detection of low-light-emitting bioluminescent dinoflagellate species and will have applications as robust indicators of dinoflagellate bioluminescence in natural water samples. [source] Expression of ,re,y luciferase gene in Erwinia amylovoraLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 4 2003Giovanna Gentilomi Abstract In this study we describe an ef,cient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the ,re,y luciferase gene of Photinus pyralis, which is further controlled by IPTG-inducible promoter. Stably transformed E. amylovora cells maintain the same infectivity as the wild-type strain and, after induction with IPTG, produce luciferase. Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL). The transformed E. amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post-infection. Our ,ndings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments. [source] The chemopreventive compound curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1 transcription in Raji DR-LUC cells,MOLECULAR CARCINOGENESIS, Issue 3 2002Manfred Hergenhahn Abstract To characterize the effects of inhibitors of Epstein-Barr virus (EBV) reactivation, we established Raji DR-LUC cells as a new test system. These cells contain the firefly luciferase (LUC) gene under the control of an immediate-early gene promoter (duplicated right region [DR]) of EBV on a self-replicating episome. Luciferase induction thus serves as an intrinsic marker indicative for EBV reactivation from latency. The tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA) induced the viral key activator BamH fragment Z left frame 1 (BZLF1) protein ("ZEBRA") in this system, as demonstrated by induction of the BZLF1 protein-responsive DR promoter upstream of the luciferase gene. Conversely, both BZLF1 protein and luciferase induction were inhibited effectively by the chemopreventive agent curcumin. Semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) further demonstrated that the EBV inducers TPA, sodium butyrate, and transforming growth factor-, (TGF-,) increased levels of the mRNA of BZLF1 mRNA at 12, 24, and 48 h after treatment in these cells. TPA treatment also induced luciferase mRNA with similar kinetics. Curcumin was found to be highly effective in decreasing TPA-, butyrate-, and TGF-,-induced levels of BZLF1 mRNA, and of TPA-induced luciferase mRNA, indicating that three major pathways of EBV are inhibited by curcumin. Electrophoretic mobility shift assays (EMSA) showed that activator protein 1 (AP-1) binding to a cognate AP-1 sequence was detected at 6 h and could be blocked by curcumin. Protein binding to the complete BZLF1 promoter ZIII site (ZIIIA+ZIIIB) demonstrated several specific complexes that gave weak signals at 6 h and 12 h but strong signals at 24 h, all of which were reduced after application of curcumin. Autostimulation of BZLF1 mRNA induction through binding to the ZIII site at 24 h was confirmed by antibody-induced supershift analysis. The present results confirm our previous finding that curcumin is an effective agent for inhibition of EBV reactivation in Raji DR-CAT cells (carrying DR-dependent chloramphenicol acetyltransferase), and they show for the first time that curcumin inhibits EBV reactivation mainly through inhibition of BZLF1 gene transcription. © 2002 Wiley-Liss, Inc. [source] Expressed Sequence Tag Analysis of the Dinoflagellate Lingulodinium polyedrum During Dark Phase,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2004Naomi Tanikawa ABSTRACT To collect information on gene expression during the dark period in the luminous dinoflagellate Lingulodinium polyedrum, normalized complementary DNA (cDNA) libraries were constructed from cells collected during the first hour of night phase in a 12:12 h light-dark cycle. A total of 4324 5,-end sequence tags were isolated. The sequences were grouped into 2111 independent expressed sequence tags (EST) from which 433 groups were established by similarity searches of the public nonredundant protein database. Homology analysis of the total sequences indicated that the luminous dinoflagellate is more similar to land plants and animals (vertebrates and invertebrates) than to prokaryotes or algae. We also isolated three bioluminescence-related (luciferase and two luciferinbinding proteins [LBP]) and 37 photosynthesis-related genes. Interestingly, two kinds of LBP genes occur in multiple copies in the genome, in contrast to the single luciferase gene. These cDNA clones and EST sequence data should provide a powerful resource for future genome-wide functional analyses for uncharacterized genes. [source] Nrf2-mediated induction of detoxifying enzymes by alantolactone present in Inula heleniumPHYTOTHERAPY RESEARCH, Issue 11 2008Ji Yeon Seo Abstract Our previous study showed that a methanol extract of Inula helenium had the potential to induce detoxifying enzymes such as quinone reductase (QR) and glutathione S -transferase (GST) activity. In this study the methanol extract was further fractionated using silica gel chromatography and vacuum liquid chromatography, to yield pure compounds alantolactone and isoalantolactone as QR inducers. Alantolactone caused a dose-dependent induction of antioxidant enzymes including QR, GST, , -glutamylcysteine synthase, glutathione reductase, and heme oxygenase 1 in hepa1c1c7 mouse hepatoma cells. The compound increased the luciferase activity of HepG2-C8 cells, transfectants carrying antioxidant response element (ARE)-luciferase gene, in a dose-dependent manner, suggesting ARE-mediated transcriptional activation of antioxidant enzymes. Alantolactone also stimulated the nuclear accumulation of Nrf2 that was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors. In conclusion, alantolactone appears to induce detoxifying enzymes via activation of PI3K and JNK signaling pathways, leading to translocation of Nrf2, and subsequent interaction between Nrf2 and ARE in the encoding genes. Copyright © 2008 John Wiley & Sons, Ltd. [source] Sequence-specific inhibition of RNA polymerase III-dependent transcription using Zorro locked nucleic acid (LNA)THE JOURNAL OF GENE MEDICINE, Issue 1 2008Rongbin Ge Abstract Background RNA polymerase III (pol III)-dependent transcripts are involved in many fundamental activities in a cell, such as splicing and protein synthesis. They also regulate cell growth and influence tumor formation. During recent years vector-based systems for expression of short hairpin (sh) RNA under the control of a pol III promoter have been developed as gene-based medicines. Therefore, there is an increasing interest in means to regulate pol III-dependent transcription. Recently, we have developed a novel anti-gene molecule ,Zorro LNA (Locked Nucleic Acid)', which simultaneously hybridizes to both strands of super-coiled DNA and potently inhibits RNA polymerase II-derived transcription. We have now applied Zorro LNA in an attempt to also control U6 promoter-driven expression of shRNA. Methods In this study, we constructed pshluc and pshluc2BS plasmids, in which U6 promoter-driven small hairpin RNA specific for luciferase gene (shluc) was without or with Zorro LNA binding sites, respectively. After hybridization of Zorro LNA to pshluc2BS, the LNA-bound plasmid was cotransfected with pEGFPluc into mammalian cells and into a mouse model. In cellular experiments, cotransfection of unhybridized pshluc2BS, Zorro LNA and pEGFPluc was also performed. Results The results showed that the Zorro LNA construct efficiently inhibited pol III-dependent transcription as an anti-gene reagent in a cellular context, including in vivo in a mouse model. Conclusions Thus, this new form of gene silencer ,Zorro LNA' could potentially serve as a versatile regulator of pol III-dependent transcription, including various forms of shRNAs. Copyright © 2007 John Wiley & Sons, Ltd. [source] Optimization of short-term transgene expression by sodium butyrate and ubiquitous chromatin opening elements (UCOEs)THE JOURNAL OF GENE MEDICINE, Issue 8 2007Jolanda J. de Poorter Abstract Background Predictable and adequate transgene expression is essential for clinical gene therapy. Several studies have focused on optimization of transgene expression. In this study the effect of sodium butyrate (NaB) and a ubiquitous chromatin opening element (UCOE) on short-term gene expression after adenovirus-mediated gene transfer in fibroblastic interface cells from periprosthetic tissue in loosened orthopedic implants is investigated. Methods Cultures of diploid human interface cells from four patients were infected with an adenovirus type-5 vector that carries the luciferase gene driven by the cytomegalovirus (CMV) promoter as a reporter. In addition, viruses with a UCOE were evaluated. Twenty-four hours after infection NaB was added in concentrations of 0 to 9 mM. Luciferase activity was tested after a further 24 h. Results NaB in a concentration of 6 mM caused a 7- to 16-fold increase in reporter gene expression compared to control condition. There was no difference in reporter gene expression when cells were infected with Ad.1.5UCOE-CMV.Luc compared to Ad.CMV.Luc. A combination of NaB and a UCOE had no advantage over NaB alone. Conclusions Addition of NaB results in a marked increase in transgene expression in cultured cells. This would allow the enhancement of the expression of the transgene, without requiring a higher vector dose. Butyrate administration could not be substituted by inclusion of UCOEs in the vector. It remains to be established whether the effective concentrations of butyrate can be obtained in vivo. Copyright © 2007 John Wiley & Sons, Ltd. [source] A reporter system for the individual detection of hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced in vivoTHE PLANT JOURNAL, Issue 3 2007Ning Shao Summary A reporter system for the assay of reactive oxygen species (ROS) was developed in Chlamydomonas reinhardtii, a plant model organism well suited for the application of inhibitors and generators of various types of ROS. This system employs various HSP70A promoter segments fused to a Renilla reniformis luciferase gene as a reporter. Transformants with the complete HSP70A promoter were inducible by both hydrogen peroxide and singlet oxygen. Constructs that lacked upstream heat-shock elements (HSEs) were inducible by hydrogen peroxide, indicating that this induction does not require such HSEs. Rather, downstream elements located between positions ,81 to ,149 with respect to the translation start site appear to be involved. In contrast, upstream sequences are essential for the response to singlet oxygen. Thus, activation by singlet oxygen appears to require promoter elements that are different from those used by hydrogen peroxide. ROS generated endogenously by treatment of the alga with metronidazole, protoporphyrin IX, dinoterb or high light intensities were detected by this reporter system, and distinguished as production of hydrogen peroxide (metronidazole) and singlet oxygen (protoporphyrin IX, dinoterb, high light). This system thus makes it possible to test whether, under varying environmental conditions including the application of abiotic stress, hydrogen peroxide or singlet oxygen or both are produced. [source] Large-scale screening of Arabidopsis circadian clock mutants by a high-throughput real-time bioluminescence monitoring systemTHE PLANT JOURNAL, Issue 1 2004Kiyoshi Onai Summary Using a high-throughput real-time bioluminescence monitoring system, we screened large numbers of Arabidopsis thaliana mutants for extensively altered circadian rhythms. We constructed reporter genes by fusing a promoter of an Arabidopsis flowering-time gene , either GIGANTEA (GI) or FLOWERING LOCUS T (FT) , to a modified firefly luciferase gene (LUC+), and we transferred the fusion gene (PGI::LUC+ or PFT::LUC+) into the Arabidopsis genome. After mutagenesis with ethyl methanesulfonate, 50 000 M2 seedlings carrying the PGI::LUC+ and 50 000 carrying PFT::LUC+ were screened their bioluminescence rhythms. We isolated six arrhythmic (AR) mutants and 29 other mutants that showed more than 3 h difference in the period length or phase of rhythms compared with the wild-type strains. The shortest period length was 16 h, the longest 27 h. Five of the six AR mutants carrying PGI::LUC+ showed arrhythmia in bioluminescence rhythms in both constant light and constant dark. These five AR mutants also showed arrhythmia in leaf movement rhythms in constant light. Genetic analysis revealed that each of the five AR mutants carried a recessive mutation in a nuclear gene and the mutations belonged to three complementation groups, and at least one of which was mapped on a novel locus. Our results suggest that the three loci identified here may contain central clock or clock-related genes, at least one of which may be a novel. [source] Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type x collagen geneARTHRITIS & RHEUMATISM, Issue 1 2009Akiro Higashikawa Objective Type X collagen and runt-related transcription factor 2 (RUNX-2) are known to be important for chondrocyte hypertrophy during skeletal growth and repair and development of osteoarthritis (OA) in mice. Aiming at clinical application, this study was undertaken to investigate transcriptional regulation of human type X collagen by RUNX-2 in human cells. Methods Localization of type X collagen and RUNX-2 was determined by immunohistochemistry, and their functional interaction was examined in cultured mouse chondrogenic ATDC-5 cells. Promoter activity of the human type X collagen gene (COL10A1) was examined in human HeLa, HuH7, and OUMS27 cells transfected with a luciferase gene containing a 4.5-kb promoter and fragments. Binding to RUNX-2 was examined by electrophoretic mobility shift assay and chromatin immunoprecipitation. Results RUNX-2 and type X collagen were co-localized in mouse limb cartilage and bone fracture callus. Gain and loss of function of RUNX-2 revealed that RUNX-2 is essential for type X collagen expression and terminal differentiation of chondrocytes. Human COL10A1 promoter activity was enhanced by RUNX-2 alone and more potently by RUNX-2 in combination with the coactivator core-binding factor , in all 3 human cell lines examined. Deletion, mutagenesis, and tandem repeat analyses identified the core responsive element as the region between ,89 and ,60 bp (termed the hypertrophy box [HY box]), which showed specific binding to RUNX-2. Other putative RUNX-2 binding motifs in the human COL10A1 promoter did not respond to RUNX-2 in human cells. Conclusion Our findings indicate that the HY box is the core element responsive to RUNX-2 in human COL10A1 promoter. Studies on molecular networks related to RUNX-2 and the HY box will lead to treatments of skeletal growth retardation, bone fracture, and OA. [source] PHOSPHORUS BIOAVAILABILITY MONITORING BY A BIOLUMINESCENT CYANOBACTERIAL SENSOR STRAIN ,JOURNAL OF PHYCOLOGY, Issue 1 2002Osnat Gillor Phosphorus (P) is widely considered to be the main nutrient limiting the productivity of freshwater phytoplankton, but an assessment of its bioavailability in natural samples is highly complex. In an attempt to provide a novel tool for this purpose, the promoter of the alkaline phosphatase gene, phoA, from Synechococcus sp. PCC 7942 was fused to the luxAB luciferase genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus sp. PCC 7942 genome to yield strain APL, which emitted light when inorganic P concentrations fell below 2.3 ,M. Light emission of P-deprived cells decreased rapidly upon inorganic P readdition. The reporter was demonstrated to be a sensitive tool for monitoring the bioavailability of both inorganic and organic P sources. In water samples taken from a natural freshwater environment (Lake Kinneret, Israel), the luminescence measured correlated with total dissolved phosphate concentrations. [source] | |||||||||||||||||||||||||