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Mol. Cell. Biol. 20(14), 5216-5226. Modulation of CRX transactivation activity by phosducin isoforms 2000

Zhu, X. and Craft, C.M.

Notes: The pRL-null Vector was used as a reporter for a DLR® assay (Dual-Luciferase® Reporter Assay System). The promoter from human IRBP (interphotoreceptor retinoid binding protein) was cloned into the HindIII site of pRL-null. Transient transfections were done with 1µg of the pIRBP, 0.4µg of expression construct, and 0.2µg of pGL3-promoter as a transfection efficiency control. Activity of the Renilla luciferase was normalized to the activity of the firefly luciferase. Cells used for this assay were COS-7 and Weri-Rb-1 retinoblastoma cells. (2149)

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Mol. Cell. Biol. 20(10), 3558-3567. Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P sites that govern translational repression. 2000

Mothe-Satney, I., Yang, D., Fadden, P., Haystead, T.A. and Lawrence, J.C., Jr

Notes: The authors designed a bicistronic reporter, designated pRLIRESFL, encoding Renilla luciferase and firefly luciferase. This construct was used to investigate cap-dependent mRNA translation. pRLIRESFL directs the synthesis of an mRNA from which Renilla luciferase is translated in a manner dependent on the 5' cap and firefly luciferase is translated in a cap-independent manner through the IRES. The Renilla luciferase coding fragment was inserted into the EcoR I cloning site of p2332, a plasmid containing the CMV promoter, sequences encoding the IRES from encephalomyocarditis virus, firefly luciferase and the SV40 t-intron and polyadenylation signal. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. (2173)

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Mol. Cell. Biol. 20(14), 4990-94. Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites. 2000

Wilson, J.E., Powell, M.J., Hoover, S.E. and Sarnow, P.

Notes: Control regions of a naturally occurring dicistronic promoter from Cricket Paralysis Virus (CrPV) were cloned into a previously described dicistronic reporter vector coding for both Fluc and Rluc. The reporter was used either for in vitro translation from RiboMAX™-produced uncapped RNA (RiboMAX™ Large-Scale RNA Production System) using either an RRL or WGE system, and measuring the translation using the Dual-Luciferase® Reporter Assay System, or for transfection into SL-2 cells. RNA was also used for transfection of the SL2 cells, and luciferase activities were measured using DLR. Luciferase readings are reported as ratio of Fluc activity to Rluc activity, with the no insert vector ratio set to 1. (2147)

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J. Biol. Chem. 275, 8600-8609.. Oxidative stress disrupts glucocorticoid hormone-dependent transcription of the amiloride-sensitive epithelial sodium channel alpha-subunit in lung epithelial cells through ERK-dependnent and thioredoxin-sensitive pathways. 2000

Wang, H.-C. , Zentner, M.D., Deng, H.-T., Kim, K.-J., Wu, R., Yang, P.-C., Ann, D.K.

Notes: Addition of exogenous H2O2 to cultured A549 cells causes a rapid increase in activated Erk as judged by the Anti-ACTIVE® MAPK pAb via Western blotting. There is an inhibition of transcriptional activation of the amiloride-sensitive epithelial sodium channel α-subunit. The inhibition is reversed by 50µM U0126 MEK Inhibitor treatment 20 minutes prior to addition of H2O2 . Reporters were used for measurement of transcriptional activity and were produced in the pGL2-Promoter Vector. The reporter vectors were cotransfected into the A549 cells with the pRL-TK Vector at a 12:1 ratio. The luciferase activities were measured with the Dual-Luciferase® Reporter Assay System. Transfections were accomplished with the ProFection® Mammalian Transfection System-DEAE-Dextran. (0200)

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Blood Cells Mol. Dis. 25(14), 193-209. Activation of the delta-globin gene by the beta-globin gene CACCC motif. 1999

Ristaldi M.S., Casula S., Porcu S., Marongiu M.F., Pirastu M. and Cao, A.

Notes: These authors created mutations in the delta-globin promoter to incorporate various combinations of three elements present in the beta-globin promoter. They then examined compared the function of this chimeric promoter in Cos7, C88 mouse erythroleukemia (MEL) and K562 erythroid cell lines compared to that of the delta-globin wild type promoter.  The chimeric globin promoters drove expression of firefly luciferase. The pRL-CMV Vector was used as a transfection control at a 3:1 ratio (4 µg total).  Relative expression levels were assayed using the Dual-Luciferase® Reporter Assay System.  The authors also used a competitive transient expression assay using a single vector containing both firefly and Renilla luciferase genes under the control of separate promoters.  Again, the chimeric delta-globin promoter (driving firefly luciferase) was tested with the wild type beta-globin promoter (driving Renilla luciferase). The ratio between the two was expressed relative to expression from a wild type beta-globin promoter driving both firefly and Renilla luciferase in the same construct.  (3059)

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J. Biol. Chem. 274, 8813-8822. CREB binding protein coordinates the function of multiple transcription factors including nuclear factor I to regulated phosphoenolpyruvate carboxykinase (GTP) gene transcription. 1999

Leahy, P., Crawford, D.R., Grossman, G., Gronostajski, R.M., Hanson, R.W.

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to demonstrate a direct interaction between the transactivation domain of nuclear factor I-C (NFIC) and the CREB binding domain of the CREB-binding protein (CBP). Four different domains of the CBP were linked to the GAL4 binding domain of the pBIND Vector, and the transactivation region of the NFIC was cloned into the pACT Vector. The positive control vectors included with the system, pBIND-Id and pACT-myo Vectors, induced a 110-fold increase in luciferase activity. One construct of the CBP protein in combination with the NFIC construct induced a 70-fold increase in luciferase activity. Equal amounts of the vectors (0.6µg) were transfected into HepG2 cells. The Dual-Luciferase® Reporter Assay System was used to assess luciferase activities. (0851)

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J. Biol. Chem. 274, 2672-2681. Cross-talk between janus kinase-signal transducer and activator of transcription (JAK-STAT) and peroxisome proliferator-activated receptor- alpha (PPARalpha) signaling pathways. Growth hormone inhibition of pparalpha transcriptional activity mediated by stat5b. 1999

Zhou, Y. C. , Waxman, D. J.

Notes: The authors co-transfected COS-1 cells with a firefly luciferase reporter construct containing three tandem repeats of a PPRE as well as expression plasmids for growth hormone receptor, JAK2, STAT5a, and PPAR alpha. The pRL-TK Vector was used as a transfection control. Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. (0061)

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J. Biol. Chem. 274, 3522-3530. Distinct and common pathways in the regulation of insulin-like growth factor-1 receptor gene expression by angiotensin II and basic fibroblast growth factor. 1999

Scheidegger, K.J., Du, J., Delafontaine, P.

Notes: The authors co-transfected firefly luciferase reporter constructs with pRL-TK Vector at a ratio of 200:1 into CHO cells overexpressing the angiotensin II AT-1A receptor. Luciferase activities were assayed with the Dual-Luciferase® Reporter Assay System. (0450)

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Oncogene 18, 1-8. Endothelial receptor tyrosine kinases activate the STAT signaling pathway: Mutant Tie-2 causing venous malformations signals a distinct STAT activation response. 1999

Korpelainen, E.I., Kärkkäinen, M., Gunji, Y., Vikkula, M., Alitalo, K.

Notes: The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to produce the R849W mutation in the Tie-2 cDNA cloned into the pcDNA3.1Z+ Vector. The Dual-Luciferase® Reporter Assay System was used with the 293T cells, but no details of the transfection ratio for the firefly luciferase reporter construct and the pRL-TK Vector are provided. (0883)

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J. Immunol. 162, 4069-4078. Expression of murine IL-12 is regulated by translational control of the p35 subunit. 1999

Babik, J.M., Adams, E., Tone, Y., Fairchild, P.J., Tone, M. and Waldmann, H.

Notes: The Dual-Luciferase® Reporter Assay System was used to study a promoter as well as the 5' UTR of the p35 gene. Reporter studies were performed in the pGL3 Enhancer Vector using a 5:1 ratio of the firefly luciferase vector to the pRL-TK Vector. Studies of the 5' UTR were performed in the pGL3 Control Vector, which was transfected at a 10:1 ratio with the pRL-TK Vector. Studies were performed in A20 B cell lymphoma cell line and the RAW 264 macrophage cells. (1488)

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Mol. Cell. Biol. 19, 515-525. Fiber-type-specific transcription of the troponin I slow gene is regulated by multiple elements. 1999

Calvo, S. , Venepally, P. , Cheng, J. , Buonanno, A.

Notes: The authors used the pCAT® Basic and pGL3 Basic Vectors to assay for various slow and fast twitch type troponin I transcriptional regulatory elements. They co-transfected pGL3 Basic-derived Vectors with pRL-SV40 Vector at a ratio of 25:1 using calcium phosphate method. Assayed luciferase activities using Dual-Luciferase® Reporter Assay System in a Berthold luminometer. (1383)

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J. Immunol. 163, 4383-4391. Functional synergism of STAT6 with either NF-κB or PU.1 to mediate IL-4-induced activation of IgE germline gene transcription. 1999

Stutz, A.M., Woisetschlager, M.

Notes: Reporter studies were performed in the human EBV-negative Burkitt's lymphoma cell line DG75. A fragment of the human germline IgE promoter was subcloned into the pGL2-Basic Vector for analysis. The firefly luciferase vector was cotransfected with the pRL-TK Vector at a 20:1 ratio to provide an internal transfection control. The firefly and Renilla luciferases were detected with the Dual-Luciferase® Reporter Assay System. (0307)

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Brain Res. Mol. Brain Res. 69, 62-72. Genomic organization and regulatory elements of the rat latexin gene, which is expressed in a cell type-specific manner in both central and peripheral nervous systems. 1999

Miyasaka, N., Hatanaka, Y., Jin, M., Arimatsu, Y.

Notes: Reporter studies were performed in two modified PC12 cell lines, PC12D and PC12-22a, NS-20 neuroblastoma cells and L6 rat skeletal myoblast-like cells. Promoter constructs were made in the pGL3 Basic Vector. These constructs were cotransfected with the pRL-CMV Vector, a Renilla luciferase expression plasmid, to control for transfection efficiency. The two vectors were transfected at a 100:1 ratio, respectively. The luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. (0696)

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J. Biol. Chem. 274, 1791-1800. Identification and characterization of the cis-acting elements of the human CD155 gene core promoter. 1999

Solecki, D., Wimmer, E., Lipp, M. , Bernhardt, G.

Notes: Linker Scanning Mutants of CD155 core promoter region cloned in pGL2 Basic Vector were transfected into several cell lines with pRL-TK Vector or pRL-SV40 Vector as a transfection control at a ratio of 36:1. The authors used the Dual-Luciferase® Reporter Assay System to measure expression from mutant promoters. The Luciferase Assay System was used to measure luciferase expression in the presence of an AP1 expression plasmid. (0349)

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J. Biol. Chem. 274, 3970-3977. Identification of a functional peroxisome proliferator-responsive element in the murine fatty acid transport protein gene. 1999

Frohnert, B. I. , Hui, T. Y. , Bernlohr, D. A.

Notes: The promoter region (and 5' deletion variants thereof) was inserted upstream of the firefly luciferase gene in pGL3 Basic Vector. These constructs were co-transfected into CV-1 cells with various different PPAR isoform expression plasmids and pRL-SV40 Vector as a transfection control at a ratio of 50: 25: 1, respectively. Luciferase activities were determined using the Dual-Luciferase® Reporter Assay System . (1125)

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Proc. Natl. Acad. Sci. USA 96, 1433-1438. Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chondrocytes. 1999

Beier, F., Lee, R.J., Taylor, A.C., Pestell, R.G. , LuValle, P.

Notes: In this study, a Cyclin D1 promoter construct (driving firefly luciferase) with expression vector for ATF-2 and pRL-SV40 Vector (transfection control) were cotransfected at a 10:13:1 ratio, respectively. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. (1476)

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Science 285, 553-556. Light-dependent sequestration of TIMELESS by CRYPTOCHROME 1999

Ceriani, M.F., Darlington, T.K., Staknis, D., Mas, P., Petti, A.A., Weitz, C.J., Kay, S.A.

Notes: Most organisms display an endogenous timekeeping mechanism, or circadian clock, which consists of negative feedback loops of gene regulation that facilitate adaptation to cycles of light and darkness. In Drosophila, as well as other organisms, several of the molecules involved in sustaining this circadian clock have been identified. A gene product required for circadian photoreception has recently been identified in Drosophila, and termed crytochrome (CRY). These researchers investigated whether CRY could interact directly with the core clock proteins PERIOD (per) and TIMELESS (tim). The Drosophila cell line S2 was transiently transfected with a firefly luciferase reporter construct under control of the tim promoter, in conjunction with different combinations of constructs expressing clk, per, tim, and cry. Data was normalized to a cotransfected reporter plasmid, either the pRL-null Vector, a Renilla luciferase vector, or a beta-galactosidase expression vector, and the resulting activities measured using either the Dual-Luciferase® Reporter Assay System or the Beta-Galactosidase Enzyme Assay System. These transfection studies, along with coimmuneprecipitation assays, a yeast two-hybrid assay, and immunolocalization studies, show that CRY can block the function of PER/TIM heterodimeric complexes in a light-dependent fashion. In addition, PER/TIM and CRY influence the subcellular distribution of these protein complexes. Thus, CRY acts as a circadian photoreceptor by directly interacting with the core protein components of the circadian clock. (1354)

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J. Biol. Chem. 274, 2665-2671. OC-2, a novel mammalian member of the ONECUT class of homeodomain transcription factors whose function in liver partially overlaps with that of hepatocyte nuclear factor-6. 1999

Jacquemin, P., Lannoy, V.J., Rousseau, G.G., Lemaigre, F.P.

Notes: The authors constructed their own Renilla luciferase transfection control from pRL-null Vector by inserting pfk-2 promoter (phosphofructokinase). Co-transfected firefly luciferase reporter constructs with OC-2 or HNF-6α expression vector and Renilla luciferase control plasmid (pRL138) into rat hepatoma FTO-2B cells at a ratio of 27:1:1, respectively. Luciferase activities were measured with Dual-Luciferase® Reporter Assay System. (0967)

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J. Biol. Chem. 274, 1277-1285. Promoter characterization of the rat gene for Ca2+-binding protein regucalcin. Transcriptional regulation by signaling factors. 1999

Murata, T., Yamaguchi, M.

Notes: Reporter studies were performed in rat H4-II-E hepatoma cells. Various 5' deletion series of rat regucalcin gene promoter were prepared in the pGL3 Basic Vector. The pGL3-based constructs were cotransfected with pRL-TK Vector at a 4:1 ratio and both firefly and Renilla luciferase activities determined with the Dual-Luciferase® Reporter Assay System. All transfections were performed with the Tfx™-20 Reagent. (0632)

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Proc. Natl. Acad. Sci. USA 96, 6694-6699. Regulation of eukaryotic protein synthesis: Selective influenza viral mRNA translation is mediated by the cellular RNA-binding protein GRSF-1. 1999

Park, Y.W., Wilusz, J., Katze, M.G.

Notes: The 5' UTR of the nucleocapsid protein was placed upstream of the firefly luciferase gene of the pSP-luc+NF Fusion Vector, termed pSP-NP. Another construct was prepared from the pSP-luc+NF Vector by amplifying the Renilla luciferase gene from the pRL-CMV Vector and replacing the firefly luciferase. A mutant 5' UTR was placed into this construct and was called pSP-NP-A. The plasmids were converted to capped mRNA by in vitro transcription, and the resulting RNAs were translated in a cell-free HeLa extract (S10 extract). The activities of the two RNAs were assessed with the Dual-Luciferase® Reporter Assay System. (0549)

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Genes Dev. 13(4), 412-23. Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor. 1999

Nomura, T., Khan, M.M., Kaul, S.C., Dong, H.D., Wadhwa, R., Colmenares, C., Kohno, I. and Ishii, S.

Notes: Repression activity of the Ski component of HDAC was studied using the Dual-Luciferase® Reporter Assay System. CV-1 cells were transfected with 3µg of luciferase reporter (6 tandem repeats of the GAL4 binding site linked to the TK promoter driving luciferase), 0.33µg of the GAL4-Ski expression plasmid, and 1µg of pRL-TK. Other amounts of reporter and expression plasmid were used in other assays for transcriptional repression. (2166)

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J. Biol. Chem. 274, 503. The human copper-zinc superoxide dismutase gene (SOD1) proximal promoter is regulated by Sp1, Egr-1 and WT1 via non-canonical binding sites. 1999

Minc, E., de Coppet, P., Masson, P., Thiery, L., Dutertre, S., Amor-Guéret, M., Jaulin, C.

Notes: Reporter studies were performed in HeLa cells. Experimental constructs were assembled in pGL2 Basic Vector and co-transfected with the Renilla luciferase vector, pRL-TK Vector, at a ratio of 15:1. Transfections were accomplished by electroporation and luciferase activities were assessed with the Dual-Luciferase® Reporter Assay System. (0693)

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J. Biol. Chem. 274, 36527-36536. The seven amino acids (547-553) of rat glucocorticoid receptor required for steroid and Hsp90 binding contain a functionally independent LXXLL motif that is critical for steroid binding. 1999

Giannoukos, G., Silverstein, A.M., Pratt, W.B., Simons, S.S., Jr.

Notes: Up to three mutations were introduced into a GST-fusion of a glucocorticoid receptor with the GeneEditor™ in vitro Site-Directed Mutagenesis System. Mutations were performed in an undefined eukaryotic expression vector. The expressed proteins were used for pull-down assays. In other experiments, the ability of mutant and wildtype receptors to activate transcription were assessed with a luciferase-based assay. The wildtype or mutant constructs were co-expressed with a GRE-containing firefly luciferase construct, and the amount of firefly luciferase produced was quantified. To control for transfection efficiency, the pRL-TK Vector was transfected as well at a 5:1 ratio. Both firefly and Renilla luciferase activities were measured with the Dual-Luciferase® Reporter Assay System. (1100)

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J. Biol. Chem. 274, 22437-22444. TIP49b, a new RuvB-like DNA helicase, is included in a complex together with another RuvB-like DNA helicase, TIP49a. 1999

Kanemaki, M., Kurokawa, Y., Matsu-ura, T., Makino, Y., Masani, A., Okazaki, K., Morishita, T., Tamura, T.

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to demonstrate an interaction between TIP49a and TIP49b in vivo. TIP49a or TIP49b were placed into the pBIND and pACT Vectors, and 50ng of each transformed into HeLa cells with 200ng of the pG5luc Vector. Both TIP49a and TIP49b were placed into the pBIND Vector and both were placed into the pACT Vector, so that all combinations of the constructs could be tested. The pBIND-TIP49b construct with the pACT-TIP49a construct produced a 15-fold increase in luciferase activity. The pBIND-TIP49a and pACT-TIP49b produced a 22-fold increase. TIP49b in both the pBIND and pACT Vectors produced only a 6-fold increase, and TIP49a in both vectors produced only a 2-fold increase. The increase in luciferase activity was in relation to the pBIND and pACT Vectors alone. The positive control vectors included in the system, pACT-MyoD Vector and the pBIND-Id Vector, produced a 221-fold increase. All firefly luciferase activities from the pG5luc Vector were normalized to the Renilla luciferase activity coming from the pBIND Vector, and both luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. (0958)

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J. Biol. Chem. 273, 14002-14007. 4E-BP3, a new member of the eukaryotic initiation factor 4E-binding protein family. 1998

Poulin, F., Gingras, A.C., Olsen, H., Chevalier, S., Sonenberg, N.

Notes: The authors created a bicistronic vector with a CMV-driven expression of Renilla luciferase followed by the IRES sequence from the 5'-UTR of poliovirus type II Lansing and trailed by the wildtype firefly luciferase gene. The construct was cotransfected into HeLa cells with CMV-driven expression vectors containing wildtype eIF-4E binding protein or mutants. The wildtype eIF-4E binding protein reduced Renilla luciferase activity by about 75% but resulted in a two-fold increase in firefly luciferase. Luciferase activities were measured with the Dual-Luciferase® Reporter Assay System and the pGEM®-luc Vector was the source of the Firefly luciferase and the pRL-CMV Vector was the source of Renilla luciferase. (0544)

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