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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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J. Immunol. 161, 3719-3728. A complex element regulates IFN-gamma-stimulated monocyte chemoattractant protein-1 gene transcription. 1998

Valente, A. J., Xie, J. F. , Abramova, M. A., Wenzel, U. O., Abboud, H. E., Graves, D. T.

Notes: The authors transfected MG-63 human osteoblastic cells with a 10:1 ratio of the MCP-1/firefly luciferase plasmid, derived from the pGL2-Basic Vector, and the pRL-CMV Vector as a control. The calcium phosphate method of transfection was used. Luciferase activities were assayed with the Dual-Luciferase® Reporter Assay System. (0213)

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J. Biol. Chem. 273, 18743-18750. A tripartite array of transcription factor binding sites mediates cAMP induction of phosphoenolpyruvate carboxykinase gene transcription and its inhibition by insulin. 1998

Yeagley, D., Agati, J.M., Quinn, P.G.

Notes: The H4eII cell line was transfected with 20µg of a pGL3 Basic Vector-derived construct, 2µg of various expression vectors and 2µg of the pRL-SV40 Vector. One of the pGL3 Basic Vector-based constructs had the PEPCK promoter plus a GAL4 binding domain and another had five tandem repeats of the GAL4 domain and a minimal TATA box. Fusion proteins of the Kinase-inducible domain and the GAL4 binding domain as well as the Kinase-inducible domain and a cAMP inducible domain. The system was used to measure activation of luciferase via phosphorylation of the GAL4 fusion protein producing a functional activation domain. All transfections and luciferase activities were normalized to Renilla luciferase activity using the Dual-Luciferase® Reporter Assay System. (0111)

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Circulation 98, 1164-1171. Activated platelets induce monocyte chemotactic protein-1 secretion and surface expression of intercellular adhesion molecule-1 on endothelial cells. 1998

Gawaz, M., Neumann, F.-J., Dickfeld, T., Koch, W., Laugwitz, K.-L., Adelsberger, H., Langenbrink, K., Page, S., Neumeier, D., Schömig, A., Brand, K.

Notes: Luciferase reporter studies were performed in primary HUVECs. Experimental construct were prepared in either pGL2 Basic or pGL3 Promoter Vector and cotransfected with the pRL-TK Vector. Reporter activity was monitored with the Dual-Luciferase® Reporter Assay System. (1147)

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J. Immunol. 161, 277-285. An NFAT-dependent enhancer is necessary for anti-IgM-mediated induction of murine CD5 expression in primary splenic B cells. 1998

Berland, R. and Wortis, H.H.

Notes: Reporter assays were performed in murine splenic B cells. Firefly luciferase reporter constructs were contransfected with an equal amount of the pRL-TK Vector. Luciferase activities were assessed with the Dual-Luciferase® Reporter Assay System. (1439)

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J. Biol. Chem. 273, 18751-18759. Assessment of the roles of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B and protein kinase C in insulin inhibition of cAMP-induced phosphoenolpyruvate carboxykinase gene transcription. 1998

Agati, J.M., Yeagley, D. and Quinn, P.G.

Notes: Up to four plasmids were transfected into H4IIe cells including 20µg of luciferase reporter vector, 2µg of pRL-SV Vector and an RSV-driven expression vector expressing the catalytic domain of PKA or ras pathway mutants. Luciferase activity was normalized to Renilla luciferase activity using the Dual-Luciferase® Reporter Assay System. (2063)

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Oncogene 16, 423-428. C-Myc 5' untranslated region contains an internal ribosome entry segment. 1998

Stoneley, M., Paulin, F. E., Le Quesne, J. P., Chappell, S. A., Willis, A. E.

Notes: The authors used the Dual-Luciferase® Reporter Assay System, pGL3 Control Vector, pRL-CMV Vector, pSV-beta-Galactosidase Control Vector, Luciferase Assay System and RNase ONE® Ribonuclease in their studies. The objective of the paper was to demonstrate that the 5' untranslated region (UTR) of c-myc functions as an internal ribosome entry site (IRES). To directly assay the ability of the UTR to function as a IRES, the Renilla luciferase gene was subcloned in front of the UTR. Dual expression of the luciferases was demonstrated in both HeLa cells and HepG2 cells and dependent upon the c-myc UTR. Mutation of the Renilla luciferase sequence to contain a hairpin structure, demonstrate that the Renilla luciferase activity could reduced by 75% with no effect on the firefly luciferase activity. RNase protection assays with a 624 nucleotide probe that crossed the Renilla luciferase cDNA, the c-myc UTR and the firefly luciferase cDNA demonstrated that only one single message is produced by the transfected HeLa cells and the c-myc UTR is not functioning as a promoter. All transfections were normalized to control β-galactosidase activity. (0339)

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Immunity 9, 247-256. CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching in non-antigen-selected human B cells 1998

Cerutti, A., Schaffer, A., Shah, S., Zan, H., Liou, H.-C., Goodwin, R.G., Casali, P.

Notes: Luciferase studies were performed in the human B cell line, CL-01. Cells were electroporated with a 40µl DNA-TE buffer solution containing 20µg of constructs in either pGL3 Basic or pGL3 Promoter Vectors and 10ng of the pRL-CMV Vector. Thus, a 1:2000 ratio of firefly to Renilla luciferase vector is achieved and activity was measured with the Dual-Luciferase® Reporter Assay System. (1353)

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J. Biol. Chem. 273, 27420-27429. Characterization of the muring fatty acid transport protein gene and its insulin response sequence. 1998

Hui, T.Y., Frohnert, B.I., Smith, A.J., Schaffer, J.E., Bernlohr, D.A.

Notes: Luciferase reporter constructs were cotransfected into 3T3-L1 preadipocytes with the pRL-CMV Vector at a 25:1 ratio and luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. RT-PCR was accomplished with AMV Reverse Transcriptase and Taq DNA Polymerase. RNase Protection Assay were performed with the aid of the RNase ONE™ Ribonuclease. (0988)

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Am. J. Physiol. 274, F753-F761. Cis- and trans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity. 1998

Miyakawa, H., Woo, S.K., Chen, C.P., Dahl, S.C., Handler, J.S., Kwon, H.M.

Notes: Use the Dual-Luciferase® Reporter Assay System to study regulatory elements from the BGT1 gene. The vectors used for these studies were the pGL2 Basic and pGL2 Promoter Vectors as primary reporters and the pRL-CMV Vector as a transfection control. (0695)

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J. Biol. Chem. 273, 33885-33888. Cloning and characterization of the 5´-flanking region of the human growth hormone secretagogue receptor gene. 1998

Kaji, H., Tai, S., Okimura, Y., Iguchi, G., Takahashi, Y., Abe, H., Chihara, K.

Notes: Inserted promoter region and its deletion variants into pGL3-Basic Vector and co-transfected with pRL-CMV Vector (no ratio given). Measured luciferase activities with Dual-Luciferase® Reporter Assay System. (0952)

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J. Biol. Chem. 273, 33848-33855. Cloning and characterization of the human β4-integrin gene promoter and enhancers. 1998

Takaoka, A.S., Yamada, T., Gotoh, M., Kanai, Y., Imai, K., Hirohashi, S.

Notes: The authors performed DNaseI footprinting using the Core Footprinting System and nuclear extract or recombinant human AP-1 (c-jun). Promoter and enhancer elements of the human β4 integrin gene were analyzed using the Dual-Luciferase® Reporter Assay System. pRL-TK Vector was used as a transfection control. The transfection ratio of pGL3-Basic-derived plasmids to pRL-TK Vector was 10:1. (0296)

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J. Biol. Chem. 273, 25472–25479. Cloning and characterization of the human PAX2 promoter. 1998

Stayner, C.K., Cunliffe, H.E., Ward, T.A. and Eccles, M.R.

Notes: MDCK and 293 cells were previously shown to express PAX2 endogenously. Transfections were carried out using FuGENE® 6 Transfection Reagent with 10% serum without antibiotics as follows. Cells (COS-7, NIH-3T3, 293 and MDCK cell lines; 5 × 105) were seeded into 60mm dishes and transfected at approximately 50–60% confluency, 16–18 hours later. Cells were harvested 24 hours after transfection. A total of 2.05μg of DNA was used in transfections to analyze the PAX2 promoter deletion series of constructs. These reactions contained 2μg of PAX2 promoter-reporter firefly luciferase plasmid (pGL2-Basic Vector) plus 0.05μg of internal control Renilla luciferase plasmid (pRL-TK Vector). For cotransfection experiments, a total of 4.05μg of DNA was used. The reactions contained 1μg of PAX2 promoter-reporter firefly luciferase plasmid and either 3μg of pBluescript DNA or 3μg of WT1 expression plasmid, plus 0.05μg of internal control Renilla luciferase plasmid (pRL Control Vector).

The DNA solutions were combined at a 1:3 ratio with FuGENE® 6 reagent (e.g. 4.05 μg of DNA to 12 μl of FuGENE® 6 reagent for transfection of each cell line. The cells were overlaid with a final 2ml of medium, for 24 hours prior to lysis. Cell lysates were assayed for luciferase production using a Dual-Luciferase® Assay System.  Luminescence was measured with a luminometer. (4302)

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J. Biol. Chem. 273, 25472-25479. Cloning and characterization of the human PAX2 promoter 1998

Stayner, C.K., Cunliffe, H.E., Ward, T.A., Eccles, M.R.

Notes: Promoter functions were tested in NIH 3T3, COS-7, 293 and MDCK cells. Experimental constructs were prepared in the pGL2 Basic Vector and cotransfected with the pRL-TK Vector at a 40:1 ratio. Luciferase activities were monitored with the Dual-Luciferase® Reporter Assay System. (0332)

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