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J. Gen. Virol. 76, 2933-2940. Identification and characterization of an early gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus. 1995

Bischoff, D.S. and Slavicek, J.M.

Notes: The G22 gene from Lymantria dispar was cloned, sequenced and expressed in the TNT® Coupled Reticulocyte Lysate System. The predicted size of the protein was in agreement with that of the in vitro generated protein. The protein has no significant sequence homology. (1415)

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Biochem. J. 306, 473-479. Identification and characterization of DNA elements implicated in the regulation of CYP4A1 transcription. 1995

Aldridge, T., Tugwood, J.D. and Green, S.

Notes: Peroxisome proliferator-activated receptor-alpha (PPARalpha) and retinoid-X receptor-alpha (RXRalpha) were synthesized using the TNT® Coupled Reticulocyte Lysate System. These proteins were used in gel retardation assays with two possible peroxisome proliferator response elements (PPREs). Element II is a functional PPRE, and both elements were responsive to activated RXRalpha. Neither RXRalpha nor PPARalpha were able to bind the elements alone, but as a heterodimer they were able to bind element II or both elements I and II. There was no binding by the heterodimer to element I in the absence of element II observed. (1825)

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J. Biol. Chem. 270, 5434-5440. Identification of human TR2 orphan receptor response element in the transcriptional initiation site of the simian virus 40 major late promoter. 1995

Lee, H. and Chang, C.

Notes: TR2 orphan receptor, a steroid-thyroid hormone receptor, was produced in vitro using the TNT® Coupled Reticulocyte Lysate System and used in EMSAs with the TR2RE-SV40 DNA response element. These results suggest that the TR2 orphan receptor is a candidate that binds to the TR2RE-SV40 region and may play some role in SV40 gene expression. (1807)

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Plant Mol. Biol. 28, 369-380. Isolation and characterisation of cDNA clones representing the genes encoding the major tuber storage protein (dioscorin) of yam (Dioscorea cayenensis Lam.). 1995

Conlan, R. S. , Griffiths, L. A. , Napier, J. A. , Shewry, P. R. , Mantell, S. , Ainsworth, C.

Notes: The cDNA for dioscorin was expressed using the TNT® Coupled Reticulocyte Lysate System. When translation was carried out in the presence of canine microsomes, the translation efficiency was increased and a band of faster mobility was produced. Dioscorin may be cotranslationally processed by cleavage of a signal peptide; in addition, other post-translational modifications (i.e., glycosylation) may occur with this protein. (1273)

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J. Biol. Chem. 270, 21765-21771. Isolation of cDNA clones encoding eight different human G protein gamma subunits, including three novel forms designated the gamma4, gamma10 and gamma11 subunits. 1995

Ray, K., Kunsch, C., Bonner, L.M., and Robishaw, J.D.

Notes: The gamma2, gamma4, gamma10 and gamma11 subunits of human G protein were transcribed and translated using the TNT® Coupled Reticulocyte Lysate System. Prenylation was examined for these subunits using [3H]farnesyl pyrophosphate or [3H]geranylgeranyl pyrophosphate. The gamma11 subunit is modified by a farnesyl group whereas gamma4 and gamma10 subunits are modified by a geranylgeranyl group. For tryptic proteolysis assays, beta and gamma subunits were cotranscribed and cotranslated in the TNT® Coupled Reticulocyte Lysate System. The cotranslation products were digested with trypsin. A protected fragment indicated beta-gamma dimers. (1786)

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Nature 375, 812-815. Mechanism of active transcriptional repression by the retinoblastoma protein. 1995

Weintraub, S.J., Chow, K.N., Luo, R.X., Zhang, S.H., He, S. and Dean, D.C.

Notes: [35S]labeled PU.1,PU.1-M, c-myc and Elf-1 proteins were synthesized using the TNT® Coupled Reticulocyte Lysate System. c-myc, Elf-1 and PU.1 each bound specifically to Rb in the E2F-1-Rb complex. The PU.1 and PU.1-M proteins were incubated with GST-E2F-1 (that had been preincubated with His-Rb to form an E2F-Rb complex). Rb-PU.1 interacts efficiently with the E2F-1/Dp-1/DNA complex. The PU.1 and PU.1-M proteins also were incubated with GST-TBP in the presence or absence of His-Rb. Rb also blocked the interaction of PU.1 with TBP. (1836)

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J. Biol. Chem. 270, 29439-29446. Molecular cloning, expression and characterization of podocalyxin-like protein 1 from rabbit as a transmembrane protein of glomerular podocytes and vascular endothelium. 1995

Kershaw, D.B., Thomas, P.E., Wharram, B.L., Goyal, M., Wiggins, J.E., Whiteside, C.I. and Wiggins, R.C.

Notes: Reactions were performed in 25µl reactions with or without 1.5µl of microsomes. The 551 residue protein had a putative signal sequence and N-linked glycosylation sequences. Expression in the presence of the microsomes did produce an increase in molecular weight. (1623)

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J. Virol. 70, 1695-1705. Molecular genetic analysis of Epstein-Barr virus Cp promoter function. 1995

Evans, T., Farrell, P.J., and Swaminathan, S.

Notes: RBP-Jk protein was transcribed and translated in vitro using the TNT® Coupled Reticulocyte Lysate System. The protein was labeled with [35S] and used in mobility shifts with PCR-generated probes to demonstrate the binding of the RBP-Jk protein to the RBP-Jk site in the Cp promoter. (1800)

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J. Cell Biol. 131, 817-831. Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus. 1995

Bohmann, K., Ferreira, J.A. and Lamond, A.I.

Notes: [35S]Methionine-labeled RalGDS (guanine nucleotide dissociation stimulator) was transcribed and translated in the TNT® T7 Coupled Reticulocyte Lysate System. The lysate was incubated with GTPase samples and used in immunoprecipitation assays with anti-RalGDS antisera. GST-R-ras, GST-H-ras and GST-Rap (Ras-like GTPases) bind full length RalGDS in a GTP-dependent manner. In addition, Raf and RalGDS compete for binding to Ras-like GTPases. (1422)

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J. Virol. 69, 3369-3380. Mutations in the DNA-binding and dimerization domains of v-Rel are responsible for altered κ B DNA-binding complexes in transformed cells. 1995

Hrdlickova, R., Nehyba, J., Bose, H.R., Jr.

Notes: The RNA-1 from tomato black ring nepovirus encodes a 250K polyprotein that is self-cleaved when synthesized in the TNT® Coupled Reticulocyte Lysate System. The polypeptides synthesized from synthetic transcripts that correspond to different proteins encoded by RNA-1 are only cleaved when they include the 23K protease. The VpgProPol and Pro Pol can also cleave the RNA-2 encoded 150K polyprotein in trans. (1016)

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Hum. Mutat. 6, 126-135. Protein truncation test: analysis of two novel point mutations at the carboxy-terminus of the human dystrophin gene associated with mental retardation. 1995

Tuffery, S. , Lenk, U. , Roberts, R. G. , Coubes, C. , Demaille, J. , Claustres, M.

Notes: RT-PCR and PTT were used to screen for mutations associated with mental retardation in DMD patients in the 3' coding region of the dystrophin gene. The TNT® T7 Coupled Reticulocyte Lysate System was used for in vitro transcription/translation reactions. (0244)

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Nat. Genet. 10, 208-212. Rapid detection of BRCA1 mutations by the protein truncation test. 1995

Hogervorst, F.B., Cornelis, R.S., Bout, M., van Vliet, M., Oosterwijk, J.C., Olmer, R., Bakker, B., Klijn, J.G., Vasen, H.F., Meijers Heijboer, H.

Notes: RT-PCR and the protein truncation test (PTT) were used to detect BRCA1 mutations. RT-PCR products were used as templates for protein synthesis in the TNT® T7 Coupled Reticulocyte Lysate System. The products were analyzed by SDS-PAGE. Eight predisposing mutations were identified. (1053)

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Biotechniques 18, 244-248. Rapid screening of open reading frames by protein synthesis with an in vitro transcription and translation assay. 1995

Switzer, W.M., Heneine, W.

Notes: This article details a method for using the TNT® Coupled Reticulocyte Lysate System to rapidly screen ORFs (using 1/2 the reaction components per assay) directly from bacterial colonies without performing plasmid preps. (0286)

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DNA Cell Biol. 14, 681-688. Role of the transcription factor C/EBP-beta in expression of a rat pregnancy-specific glycoprotein gene. 1995

Chen, H., Lin, B., Chen, C.L., Johnson, P.F., Chou, J.Y.

Notes: Gel mobility-shift assays were performed using C/EBP synthesized in vitro using the TNT® Coupled Reticulocyte Lysate System. The DNA probe was part of the 5´ flanking region of the rodent pregnancy-specific glycoprotein gene, rnCGM3. The C/EBP proteins bind to the tested DNA fragment. (1829)

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Mol. Cell. Biol. 15, 4727-4734. Ser-3 is important for regulating Mos interaction with and stimulation of mitogen-activated protein kinase kinase. 1995

Chen, M. , Cooper, J. A.

Notes: [35S]methionine-labeled RalGDS (guanine nucleotide dissociation stimulator) was transcribed and translated in the TNT® T7 Coupled Reticulocyte Lysate System. The lysate was incubated with GTPase samples and used in immunoprecipitation assays with anti-RalGDS antisera. GST-R-ras, GST-H-ras and GST-Rap (Ras-like GTPases) bind with full length RalGDS in a GTP-dependent manner. (1327)

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J. Biol. Chem. 270, 30129-30133. Suppression of gene expression on the simian virus 40 major late promoter by human TR4 orphan receptor. 1995

Lee, H.J., Lee, Y., Burbach, J.P. and Chang, C.

Notes: Full-length and truncated TR4 orphan receptor were transcribed and translated in the TNT® Coupled Reticulocyte Lysate System. The full-length protein was used in in vitro binding assays with the +55 region of the SV40-MLP and was shown to bind specifically. In analyses of the domain features of TR4, the original DNA-protein complexes could only be detected in supershifts of C-terminal or N-terminal truncations where the proteins were incubated with the full-length TR4 and DNA probe. (1857)

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Development 121, 2667-2679. The Drosophila 63F early puff contains E63-1, an ecdysone-inducible gene that encodes a novel Ca(2+)-binding protein. 1995

Andres, A.J. and Thummel, C.S.

Notes: E63-1, CaM (positive control) and USP (negative control) were transcribed and translated using the TNT® Coupled Reticulocyte Lysate System. Labeled proteins were dialyzed against binding buffer, incubated in either EGTA or CaCl2 and run on SDS-PAGE gels. The results show that E63-1 binds Ca2+. (1513)

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Am. J. Hum. Genet. 57, 311-320. The identification of point mutations in Duchenne Muscular Dystrophy patients by using reverse-transcription PCR and the protein truncation test. 1995

Gardner, R.J., Bobrow, M. and Roberts, R.G.

Notes: The protein truncation test (PTT) was used to monitor the integrity of the dystrophin ORF in DMD patients. The TNT® T7 Coupled Reticulocyte Lysate System was used for PTT analysis in the presence of [35S]methionine. (In ~66% of DMD patients, a deletion in dystrophin is the causative agent.) Discontinuous SDS-PAGE is used to separate the proteins produced, and individuals carrying certain mutations (e.g., nonsense) are easily detected. (1870)

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Cell 81, 1105-1114. The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication. 1995

Kaufman, P.D., Kobayashi, R., Kessier, N., and Stillman, B.

Notes: p150 and p60 proteins were produced using the TNT® Coupled Reticulocyte Lysate System. Wild type and mutant p150 proteins were checked for chromatin assembly and coprecipitation with p60 using the anti-p60 antibody. The p60 proteins were mixed with GST-p150 fusion proteins and precipitated with glutathione-CL4B resin. Detection of p60 was performed with anti-p60. A direct interaction was shown for p60 and p150. (1768)

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Proc. Natl. Acad. Sci. USA 92, 3115-3119. The retinoblastoma-susceptibility gene product binds directly to the human TATA-binding protein-associated factor TAFII250. 1995

Shao, Z., Ruppert, S., Robbins, P.D.

Notes: [35S]-labeled TAFs were transcribed and translated using the TNT® T7 or T3 Coupled Reticulocyte Lysate System. These proteins were used in GST-RB binding assays, and found to bind GST-RB. This interaction suggests the potential of RB binding to the TAFs and stimulating Sp1-mediated transcription. (0428)

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Proc. Natl. Acad. Sci. USA 92, 1535-1539. Transcription factor TFIIB and the vitamin D receptor cooperatively activate ligand-dependent transcription. 1995

Blanco, J.C., Wang, I.M., Tsai, S.Y., Tsai, M.J., O'Malley, B.W., Jurutka, P.W., Haussler, M.R. and Ozato, K.

Notes: The hVDR (human 1,25 dihydroxyvitamin D3 receptor) in pSG5 was used for transfection and preparation of in vitro translated receptor. The TNT® Coupled Reticulocyte Lysate System was used to transcribe and translate the pSG5-hVDR DNA after digestion with appropriate restriction enzymes. hVDR specifically binds to TFIIB in glutathione S-transferase fusion-based protein-protein binding assays. (2240)

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Virology 208, 215225. Translation of the second gene of peanut clump virus RNA 2 occurs by leaky scanning in vitro. 1995

Herzog, E., Guilley, H. and Fritsch, C.

Notes: The initation of translation of the second AUG of RNA 2 was studied using run-off transcription in the TNT® T7 Coupled Reticulocyte Lysate System. A protein of 39kDa is produced from this RNA 2 transcript. The results ruled out mechanisms of translation initation involving termination, reinitation and internal ribosome entry. The results were consistent with a leaky scanning mechanism for translation initiation. (1905)

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J. Virol. 68, 5772-5780. A 100-kilodalton polypeptide encoded by open reading frame (ORF) 1b of the coronavirus infectious bronchitis virus is processed by ORF 1a products. 1994

Liu, D.X., Brierley, I., Tibbles, K.W., Brown, T.D.

Notes: In order to locate the region containing the 10kDa protein, proteins encoded by the IBV sequence from 10752-12600 were transcribed and translation using the TNT® T7 Coupled Reticulocyte Lysate System and immunoprecipitated with the V47 antiserum. Four protein species of approximately 38, 56, 60 and 70kDa were immunoprecipitated by antiserum V47. The results demonstrate that the antigenic determinant is encoded between nucleotides 11488 and 11739. (0773)

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Cell 76, 333-343. A novel macromolecular structure is a target of the promyelocyte-retinoic acid receptor oncoprotein. 1994

Dyck, J.A., Maul, G.G., Miller, W.H. Jr., Chen, J.D., Kakizuka, A. and Evans, R.M.

Notes: [35S]methionine-labeled PML-1 and RARalpha were synthesized in vitro using the TNT® Coupled Reticulocyte Lysate System. These proteins were used for coprecipitation experiments. Glutathione bead precipitation demonstrates that PML but not RAR can be coprecipitated with GST-PML-RAR. (1792)

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J. Biol. Chem. 269, 30069-30072. A novel RING Finger Protein Interacts with the Cytoplasmic Domain of CD40. 1994

Hu, H.M., O'Rourke, K., Boguski, M.S., Dixit, V.M.

Notes: [35S]methionine-labeled CD40bp and luciferase were prepared using the TNT® T7 Coupled Reticulocyte Lysate System. The proteins were used in interaction assays with GST fusion proteins. The results show that CD40bp only associates with GST-CD40T when the cytoplasmic domain is wild type. A mutation of Thr234 to Ala in CD40T abolishes the interaction. (1772)

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