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J. Biol. Chem. 272(33), 20800-20804. Cytosolic degradation of T-cell receptor a chains by the proteasome. 1997

Yu, H., Kaung, G., Kobayashi, S. and Kopito, R.R

Notes: The TCRa was translated using the TNT® Coupled Reticulocyte Lysate System in the presence or absence of canine pancreatic microsomes and demonstrated glycosylation of the protein. The researchers used their own preparation of microsomes made by the Walter and Blobel Protocol in Methods in Enzymology, Vol. 96, p84-93 (1983). (2045)

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J. Biol. Chem. 272, 3336-3345. Differential interaction of 1alpha,25-dihydroxyvitamin D3 analogues and their 20-epi homologues with the vitamin D receptor. 1997

Liu, Y.Y., Collins, E.D., Norman, A.W., Peleg, S.

Notes: The Altered Sites® II in vitro Mutagenesis System was used to produce several vitamin D receptor truncation mutants. The mutants were generated by the insertion of premature stop codons. The single-stranded protocol was used for the synthesis reaction and the newly synthesized DNA was propagated in BMH 71-18 mutS cells followed by growth in JM109 cells. Both mutant and wildtype receptor were cloned into pGEM®-4 Vector and in vitro expressed with the TNT® Coupled Reticulocyte Lysate System. The various forms of the receptor were assessed for protease sensitivity in the presence or absence of ligand. (0783)

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Proc. Natl. Acad. Sci. USA 94, 2563-2568. EEN encodes for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to MLL in human leukemia. 1997

So, C.W., Caldas, C., Liu, M.M., Chen, S.J., Huang, Q.H., Gu, L.J., Sham, M.H., Wiedemann, L.M. and Chan, L.C.

Notes: Promega's pGEM®-T Vector System and TNT® Coupled Reticulocyte Lysate System were used in this study. (1948)

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Cell 90, 1061-1071. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor. 1997

Kutay, U., Bischoff, F.R., Kostka, S., Kraft, R. and Görlich, D.

Notes: CAS and importin beta were cloned into the T7 60 Vector (Görlick et al. 1997), which contains a T7 promoter. The proteins were synthesized in the TNT® T7 Coupled Reticulocyte Lysate System with [35S]methionine label. The [35S]methionine-labeled proteins were incubated with importin alpha and forms of Ran. Permanently GTP-bound mutants of Ran abolish importin beta binding to alpha, but stimulate CAS-importin alpha interaction. (1824)

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Science 277, 973-974. Expression cloning in the test tube. 1997

King, R.W., Lustig, K.D., Stukenberg, P.T., McGarry, T.J., Kirschner, M.W.

Notes: This Tech. Sight article describes the in vitro expression cloning technique (IVEC). The technique involves expression of plasmid pools (~50-100 clones/pool) in the TNT® Coupled Reticulocyte Lysate System, where approximately 30 proteins can be expressed in a single reaction. Each pool is screened for a certain biochemical activity, and positive pools are progressively subdivided until a single cDNA encoding the active protein is isolated. The technique is demonstrated diagrammatically with post-translational modifications (e.g., phosphorylation, degradation and cleavage of radiolabeled protein), but other activities could be assayed with this technique. (0901)

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Nat. Genet. 17, 79-83. Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC. 1997

Laken, S.J., Petersen, G.M., Gruber, S.B., Oddoux, C., Ostrer, H., Giardiello, F.M., Hamilton, S.R., Hampel, H., Markowitz, A., Klimstra, D., Jhanwar, S., Winawer, S., Offit, K., Luce, M.C., Kinzler, K.W., Vogelstein, B.

Notes: This research was based upon the technique of Powell et al. (1993) to screen for APC mutations in Ashkenazi Jews. A mutation of APC, which carries a T-A transversion but does not have any truncating mutations, causes truncations of translation in the TNT® T7 Coupled Reticulocyte Lysate System. (0830)

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Am. J. Physiol. 273, F801-F806. Identification of a new gene product (diphor-1) regulated by dietary phosphate. 1997

Custer, M. , Spindler, B. , Verrey, F. , Murer, H. , Biber, J.

Notes: The TNT® Coupled Reticulocyte System and Canine Pancreatic Microsomal Membranes (CMMs) were used to express a clone isolated through a differential display screen. (1291)

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J. Biol. Chem. 272, 18869-18874.. Identification of CDK4 Sequences Involved in Cyclin D1 and p16 Binding 1997

Coleman, K. G. , Wautlet, B. S. , Morrissey, D. , Mulheron, J. , Sedman, S. A. , Brinkley, P. , Price, S. , Webster, K. R.

Notes: The Altered Sites® II in vitro Mutagenesis System was used to produce 27 mutant protein cDNAs. Some had up to three amino acid changes and one had a three amino acid deletion. All mutants were expressed in a TNT® Coupled Reticulocyte Lysate System reaction and the labeled proteins were used to demonstrate interactions with cyclin D1 and p16 by immunoprecipitation. (1315)

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J. Biol. Chem. 272(32), 19697-19707. Identification of membrane insertion sequences of the rabbit gastric cholecystokinin-A receptor by in vitro translation. 1997

Bayle, D., Weeks, D. and Sachs, G.

Notes: The TNT® Coupled Reticulocyte Lysate System was used to characterize the membrane domain of several polytopic proteins. The two systems employed here use the presence or inhibition of glycosylation as an indicator of membrane insertion. The addition of a core glycosylation site resulted in an increase of ~2.5kDa to the relative molecular mass determined by SDS-PAGE. Using fusion vectors HK-M0 and HK-M1, the ability of each transmembrane domain to insert into the membrane was assessed. Of seven putative membrane segments, some had signal anchor properties, one had stop transfer properties and another had no membrane insertion activity. Combining data from the different systems confirmed seven transmembrane segments. (1649)

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J. Biol. Chem. 272, 15049-15052. Identification of the MDM2 oncoprotein as a substrate for CPP32-like apoptotic proteases. 1997

Erhardt, P. , Tomaselli, K.J. , Cooper, G.M.

Notes: The Altered Sites® II in vitro Mutagenesis System was used to introduce a point mutation D359E into the MDM2 oncoprotein. Wild-type MDM2 was in vitro translated with TNT® T7Coupled Reticulocyte Lysate System and assayed for proteolysis by normal and apoptotic cell extracts. (1222)

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Proc. Natl. Acad. Sci. USA 94, 4937-4942. In vitro selection and evolution of functional proteins by using ribosome display. 1997

Hanes, J., Pluckthun, A.

Notes: [35S]methionine-labeled pre-IL-1β and PARP(T) proteins were synthesized using the TNT® T7 Coupled Reticulocyte Lysate System and were used for cleavage studies. The proteins were incubated with purified recombinant human ICE, which cleaved both proteins. The cleavage of PARP requires 50- to 100-fold higher concentrations of ICE than required for pre-IL-1β cleavage. (1060)

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J. Biol. Chem. 272, 3109-3116. Induction of the intronic enhancer of the human ciliary neurotrophic factor receptor (CNTFRalpha) gene by the TR4 orphan receptor: A member of steroid receptor superfamily. 1997

Young, W.J., Smith, S.M. and Chang, C.

Notes: The TNT® Coupled Rabbit Reticulocyte Lysate System was used for invitro translation of protein from a cDNA clone. This in vitro translated protein was used for gel shift assays and Scatchard analysis of the DNA-TR4 protein interaction. The pCAT® Promoter Vector was used to assay the enhancer activities of the intronic region. (1537)

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Cell 90, 437-447. Inscuteable and Staufen mediate asymmetric localization and segregation of prospero RNA during Drosophila neuroblast cell divisions. 1997

Li, P., Yang, X., Wasser, M., Cai, Y. and Chia, W.

Notes: The TNT® Coupled Reticulocyte Lysate System was used to generate 35S-labeled Staufen protein. The protein was used to demonstrate direct interaction between Staufen and the Inscrutable-GST fusion protein immobilized on glutathione beads. (1516)

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J. Biol. Chem. 272, 29821-29828. Interaction between the amino- and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators. 1997

Ikonen, T., Palvimo, J.J., Janne, O.A.

Notes: The authors used the Promega pSV-β-Galactosidase Control Vector, pGL3-Control and pGL3-Basic Vectors, Luciferase Assay Reagent, TNT® Coupled Wheat Germ Extract Systems and TNT® Coupled Reticulocyte Lysate Systems in their studies NF-κB. (0994)

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J. Biol. Chem. 272(19), 12667-12675. Kalirin, a cytosolic protein with spectrin-like and GDP/GTP exchange factor-like domains that interacts with peptidylglycine alpha-amidating monooxygenase, an integral membrane peptide-processing enzyme. 1997

Alam, M.R., Johnson, R.C., Darlington, D.N., Hand, T.A., Mains, R.E. and Eipper, B.A.

Notes: Poly(A+) RNA was isolated from total RNA of various rat tissues. Poly(A+) RNA was reverse transcribed with the RT System and the products used for 3'RACE. The TNT® System was used to confirm the size of the expected protein. (2140)

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EMBO J. 16, 1427-1435. Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine. 1997

White, R. , Sjoberg, M. , Kalkhoven, E. , Parker, M. G.

Notes: Wildtype mouse oestrogen receptors and various tyrosine-replacement mutants were reacted with a biotinylated oestrogen response element and immobilized on the Streptavidin MagneSphere® Paramagnetic Particles (SA-PMPs). The immobilized complexes were then assayed for the ability to interact with in vitro translated RIP140 and SRC-1 protein in the presence or absence of 17β-estradiol. The capture proteins were analyzed by SDS-PAGE and fluorography. In vitro translations were performed with the TNT® Coupled Reticulocyte Lysate System. (0181)

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EMBO J. 16, 5483-5490. Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins 1997

Zeiner, M. , Gebauer, M. , Gehring, U.

Notes: Calf Intestinal Alkaline Phosphatase was used for in vitro dephosphorylation of the RAP46 protein, and the dephosphorylated protein was assayed for interaction with various transcription factors. The TNT® Coupled Reticulocyte Lysate System was used to produce the transcription factors. The Rabbit Reticulocyte Lysate was used to aid protein refolding of heat denatured luciferase and the extent of refolding was examined with the Luciferase Assay System. (0084)

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J. Cell Biol. 138, 575-588. Molecular characterization of abLIM, a novel actin-binding and double zinc finger protein. 1997

Roof, D.J., Hayes, A., Adamian, M., Chishti, A.H. and Li, T.

Notes: Three isoforms of the actin-binding LIM protein were expressed in vitro using the TNT® Coupled Reticulocyte Lysate System in the presence of Canine Microsomal Membranes. The expressed proteins were used in an actin binding assay. (1631)

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J. Biol. Chem. 272, 30329-30333. Molecular cloning of a new aquaporin from rat pancreas and liver. 1997

Koyama, Y., Yamamoto, T., Kondo, D., Funaki, H., Yaoita, E., Kawasaki ,K., Sato, N., Hatakeyama, K. and Kihara, I.

Notes: The AQP8 protein was expressed in vitro using the TNT® Coupled Reticulocyte Lysate System in the presence of Canine Microsomal Membranes. The protein demonstrated an increase in molecular weight in the presence of the membranes. Promega's pGEM®-T Vector System and Proteinase K were also used. (1624)

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Am. J. Hum. Genet. 61, 1044-1052. Mutation detection in the repeated part of the PKD1 gene. 1997

Roelfsema, J.H., Spruit, L., Saris, J.J., Chang, P., Pirson, Y., van Ommen, G.J., Peters, D.J., Breuning, M.H.

Notes: Point mutations in the repeated portion of the PKD1 gene were detected by the Protein Truncation Test. PCR products containing the T7 promoter were used as templates and protein was labeled by incorporation of [3H] leucine in a transcription/translation reaction using TNT® Coupled Reticulocyte Lysate System. The pGEM®-T Vector System was also used. (0489)

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J. Biol. Chem. 272, 5936-5942. Nerve growth factor up-regulates the N-methyl-D-aspartate receptor subunit 1 promoter in PC12 cells. 1997

Bai, G. and Kusiak, J.W.

Notes: Reporter vectors (produced with the pGL2 Basic Vector) were transiently transfected into PC12 cells and promoter activity was measured in response to 2.5S NGF. Promoter constructs were generated by PCR, subcloned into the pGEM®-T Vector and sequenced. The pGEM®-luc Vector was used to generated an antisense luc RNA probe used in RNase protection assays (using RNase ONE™ Ribonuclease) to follow the transcription of luciferase DNA following NGF treatment. The early growth reaction transcription factor and variants were expressed in the TNT® SP6 Coupled Reticulocyte Lysate System and used to perform gel shifts with promoter elements. The SP1 Consensus Oligonucleotide was used to compete transcription factor binding to the promoter elements. (1493)

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J. Neurosci. 17, 5129-5135. NMDA receptor and the tyrosine phosphorylation of its 2B subunit in taste learning in the rat insular cortex. 1997

Rosenblum, K., Berman, D.E., Hazvi, S., Lamprecht, R. and Dudai, Y.

Notes: The NMDA subunit 2B (NR2B) and subunit 2A (NR2A) were in vitro transcribed/translated to test for specificity of an antibody raised to NR2B. The antibody precipitated the 162kDa NR2B protein but not the NR2A protein. The TNT® Coupled Reticulocyte Lysate System was used for transcription/translation. (1525)

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J. Virol. 71, 1814-1820. Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein identification of a 10-kilodalton polypeptide and determination of its cleavage sites. 1997

Liu, D.X., Xu, H.Y. and Brown, D.K.

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. (1767)

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Science 275, 1943-1947. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. 1997

Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S.I., Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., Bigner, S.H., Giovanella, B.C., Ittmann, M., Tycko, B., Hibshoosh, H., Wigler, M.H., Parsons, R.

Notes: To search for mutations in the PTEN gene, PTT was performed using the TNT® T7 Coupled Reticulocyte Lysate System, on 20 breast, 6 glioblastoma and 2 prostate tumor cell lines. Two truncating mutations were identified in PTEN in the breast samples, three in the six glioblastoma lines and in both of the prostate cancer cell lines. One of the mutations in the prostate cancer cell line affected translation initiation. It is believe that more mutations may exist in these lines but were not detected due to the limits of PTT and the fact that the entire coding region was not analyzed. (0794)

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J. Biol. Chem. 272, 23880-23886. Rat brain p64H1, expression of a new member of the p64 chloride channel protein family in endoplasmic reticulum. 1997

Duncan, R.R., Westwood, P.K., Boyd, A. and Ashley, R.H.

Notes: The pCI Mammalian Expression Vector was used to express the p64H1 protein in 293 cells and HT-4, a clonal neuronal cell line. Inserts in the vectors were verified for expression using the TNT® Coupled Reticulocyte Lysate System. The proteins were translated in the presence of Canine Pancreatic Microsomal Membranes (CMMs). The pGEM®-T Vector System was used to clone the PCR product of circle rapid amplification of cDNA ends to find the true 5'-end of the p64H1 cDNA.The Tfx™-20 Reagent was used to transfect 293 cells and HT-4 neuronal cells with the p64H1-expressing pCI-based plasmid. (1206)

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