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J. Biomol. Scr. 16(9), 995–1006. A biochemical screen for identification of small-molecule regulators of the wnt pathway using Xenopus egg extracts. 2011

Thorne, C.A., Lafleur, B., Lewis, M., Hanson, A.J., Jernigan, K.K., Weaver, D.C., Huppert, K.A., Chen, T.W., Wichaidit, C., Cselenyi, C.S., Tahinci, E., Meyers, K.C., Waskow, E., Orton, D., Salic, A., Lee, L.A., Robbins, D.J., Huppert, S.S. and Lee, E.

Notes: The authors used a biochemical assay using Xenopus egg extracts to monitor degradation levels of two Wnt pathway components, Axin and ß-catenin, and identify modulators of the Wnt pathway. ß-catenin and Axin were expressed in vitro as firefly and Renilla luciferase fusion proteins, respectively, using the TNT® SP6 High-Yield Protein Expression System and shown to behave in Xenopus extracts in a similar way to wildtype proteins, which were expressed as radiolabled proteins in rabbit reticulocyte lysate. Degradation of labeled proteins was monitored by SDS polyacrylamide gel electrophoresis and autoradiography, and degradation of luciferase fusion proteins was examined using the Dual-Glo® Luciferase Assay. Using the Xenopus extract-based assay, the authors screened chemical libraries to identify two modulators of the Wnt pathway, then confirmed this inhibition in HEK 293 cells by demonstrating a decrease in ß-catenin expression with increasing concentrations of the inhibitors. This decrease in ß-catenin-Fluc levels was measured using the Steady-Glo™ Luciferase Assay, and luciferase activity was normalized to cell viability, as determined using the CellTiter-Glo® Assay. (4181)

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J. Neuroendocrinol. 19, 309-319. Inhibition of vasopressin V1b receptor translation by upstream open reading frames in the 5'-untranslated region. 2007

Rabadan-Diehl, C., Martínez, A., Volpi, S., Subburaju, S. and Aguilera, G.

Notes: The authors studied the rat VP V1b receptor (V1bR) gene including the 826 base 5´ UTR, which has five ORFs upstream (uORF) of the V1bR protein start codon, to examine the effect of the upstream peptides on V1bR translation. The V1bR gene was cloned into the pALTER®-MAX Vector, and substitution mutations were created in the uORF translation initiation codons with the Altered Sites® II in vitro Mutagenesis System. One microgram of the linearized pALTER®- V1bR construct was transcribed using the Riboprobe® System and translated using Wheat Germ Extract with or without 35S-methionine. The unlabeled protein was analyzed by Western blot. The radiolabeled protein was spun through a 3% sucrose cushion, precipitated and separated by SDS-PAGE. For a possible membrane-targeted protein, the uORF1 was transcribed in vitro and then translated using Rabbit Reticulocyte Lysate, Canine Pancreatic Microsomal Membanes and 35S-methionine. The proteins were spun through a 3% sucrose cushion and then run on a 15% SDS-PAGE gel to determine membrane presence. (3749)

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Proc. Natl. Acad. Sci. USA 104, 66-71. Oxidized messenger RNA induces translation errors. 2007

Tanaka, M., Chock, P.B., and Stadtman, E.R.

Notes: Oxidative damage has been associated with a range of age-related neurological conditions. In this study, the effect of mRNA oxidation was investigated. A direct correlation was observed between the extent of oxidation and the frequency of translation errors. The authors excised the firefly luciferase (luc2) gene from the pGL4.14 Vector, attached a FLAG tag to the 5´ terminus and a Myc tag to the 3´ terminus, and subcloned the gene into a pGEM-4Z Vector that had been modified to append a poly(A) sequence. The construct was transfected into HEK293 cells, which were then cultured in the presence of an oxidizing agent. The occurrence of truncated protein fragments and short peptides increased in the presence of the oxidizing agent in a concentration-dependent manner. The effects of oxidation of mRNA were also investigated in in vitro translation experiments using mRNA treated with an iron-ascorbate mixture and hydrogen peroxide. Translation in vitro was performed using rabbit reticulocyte lysate supplemented with protease inhibitors. The translation products were detected using anti-FLAG and anti-c-Myc antibodies. (3630)

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Mol. Cell 22, 553-560. Recapitulation of short RNA-directed translational gene silencing in vitro. 2006

Wang, B., Love, T.M., Call, M.E., Doench, J.G. and Novina, C.D.

Notes: microRNAs (miRNAs) are a class of endogenous short RNAs that repress gene expression. To assess miRNA-directed translational silencing, in vitro reactions were performed. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a poly(A) tail. In vitro transcription was performed with 5μg of linearized plasmids containing zero, two, four, or six miCXCR4 binding sites, one siCXCR4 binding site or Renilla luciferase (pRL-TK; control mRNA) using the RiboMax™ T7 Large-Scale RNA Production kit. The mRNAs were modified by 7-methyl-G capping and polyadenylation. Translation was performed using nuclease-treated rabbit reticulocyte lysate containing 0.025pmole of mRNA with miCXCR4 or siCXCR4 binding sites, 0.025pmole Renilla control RNA, and different ratios of CXCR4 siRNA. Reaction products were separated by SDS-PAGE and visualized and quantitated by PhosphorImager analysis. (3412)

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J. Biol. Chem. 280, 41512-41520. The Cystic Fibrosis transmembrane conductance regulator is regulated by a direct interaction with the protein phosphatase 2A. 2006

Thelin, W.R., Kesimer, M., Tarran, R., Kreda, S.M., Grubb, B.R., Sheehan, J.K., Stutts, M. J. and Milgram, S.L.

Notes: This study investigated the role of phosphatases in the deactivation of the Cystic Fibrosis transmembrane conductance regulator (CFTR). CFTR C-terminal peptides were synthesized and immobilized. They were incubated with recombinant PP2A B´ε subunit produced from Rabbit Reticulocyte Lysate or BL21 cells. The recombinant B´ε subunit binds specifically to CFTR-(1451-1476) C-terminal peptide. The core PP2A subunit and A regulatory subunit do not bind to CFTR. (3528)

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J. Biol. Chem. 280, 28215-28220. Determination of the functionality of common APOA5 polymorphisms. 2005

Talmud, P.J., Palmen, J., Putt, W., Lins, L., and Humphries, S.E.

Notes: The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM®-7Zf Vector. Transcription/translation experiments were performed using the TNT® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ GreenLys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe® System –T7 and the Ribo m7G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

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J. Biomol. Scr. 9, 3-11. A strategy for discovery of novel broad-spectrum antibacterials using a high-throughput Streptococcus pneumoniae transcription/translation screen. 2004

Pratt, S.D., David, C.A., Black-Schaefer, C., Dandliker, P.J., Xuei, X., Warrior, U., Burns, D.J., Zhong, P., Cao, Z., Saiki, A.Y.C., Lerner, C.G., Chovan, L.E., Soni, N.B., Nilius, A.M., Wagenaar, F.L., Merta, P.J., Traphagen, L. and Beutel, B.A.

Notes: Several different sequences from a S. pneumoniae pA promoter region were cloned into the pSP-luc+ vector and screened for expression levels in in vitro transcription/translation systems. The RiboMAX™ SP6 Large Scale RNA Production System was used to transcribe luciferase encoding mRNAs.  Luciferase mRNAs and plasmids were used as templates in high throughput inhibition studies in an S. pneumoniae S30 extract described in the paper as well as in Promega’s E. Coli S30 Extract System for Circular DNA and in Rabbit Reticulocyte Lysate. Promega amino acid mixtures were also used in these studies. (3227)

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J. Cell Sci. 117, 2937-49. Carbohydrates act as sorting determinants in ER-associated degradation of tryosinase. 2004

Svedine, S., Wang, T., Halaban, R. and Herbert, D.N.

Notes: Wildtype and albino mutant tyrosinase were translated in Rabbit Reticulocyte Lysate, Nuclease Treated, supplemented with canine microsomal membranes or semipermeabilized melanocytes. After translation, SP-melanocytes were isolated and resuspended in Rabbit Reticulocyte Lysate, Untreated, in the presence of an ATP regenerating system, and ER-associated degradation of the proteins was evaluated. (3375)

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Proc. Natl. Acad. Sci. USA 101(9), 2794-2799. Differential utilization of upstream AUGs in the beta-secretase mRNA suggests that a shunting mechanism regulates translation. 2004

Rogers G.W. Jr, Edelman G.M. and Mauro V.P.

Notes: The authors studied the behavior of the three upstream AUGs to the initiation codon of beta-Secretase (BACE1) in a cell-free expression system to understand how this 5´ upstream region affects translation.  The 5´ UTR (with or without a hairpin structure) was cloned in front of the firefly luciferase gene derived from the pGL3-Control Vector. The resultant construct was either transfected into cells or in vitro transcribed to yield capped or uncapped RNA followed by translation in Rabbit Reticulocyte Lysate, Nuclease Treated, with or without m7GpppG cap analogue.  The experiment demonstrated that both the hairpin structure and the 5´ cap analogue decreased the translation level of the BACE1 protein.  (3076)

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RNA 8(9), 1120-1128. Characterization of a novel antibacterial agent that inhibits bacterial translation. 2002

Boddeker, N., Bahador, G., Gibbs, C., Mabery, E., Wolf, J., Xu, L. and Watson, J.

Notes: In this paper, the effect of the novel antibiotic GS7128 on translation was determined in cell-free systems. The effective concentration for inhibition of translation was determined for both prokayotes and eukaryotes by translating β-galactosidase from a pGEM® vector in E. coli S30 extracts, and luciferase control RNA in rabbit reticulocyte lysates. It was determined that GS7128 partially inhibits initiation and can fully inhibit elongation of peptides by blocking the peptidyl transferase reaction. GS7128 was shown to bind ribosomes differently than any other characterized antibiotic. GS7128 resistant mutants were not resistant to any other antimicrobial agents.  (2686)

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J. Biol. Chem. 277, 17349-17358. Structural Determinants of BRCA1 Translational Regulation 2002

Sobczak, K. and Krzyzosiak, W.J.

Notes: The authors investigated expression of BRCA1 from alternative transcripts possessing different 5' UTRs. RT-PCR using Promega's AMV reverse transcriptase was performed to generate cDNAs from total RNA isolated from human tissue. Promega's Taq DNA Polymerase was used for the PCR reaction. The authors also prepared two mRNAs that contained one or the other of the 2 alternative 5' UTRs (ex1a or ex1b) fused with the luciferase coding region. To generate the corresponding DNA for these constructs, the luciferase coding region was amplified from Promega's pGEM vector and ligated to PCR amplified ex1a or exlb. These ligation products served as the template amplifying two cDNA constructs (exla-luc or ex1b-luc).Ten additional cDNAs were made containing a variety of changes to the 5'UTR region of BRCA1. In vitro translation experiments were performed to determine how the composition of the 5'UTR affected protein expression levels (using Promega's RNasin to protect transcribed mRNA; T7 polymerase buffer; and rabbit reticulocyte and wheat germ extracts for translation). The authors conclude that secondary structures associated with elements in the longer 5'UTR reduced translation rates and could be responsible for the reduced expression of BRCA1 often associated with spontaneous ovarian and breast cancers. (2441)

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Mol. Cell. Biol. 20(10), 3510-3521. Facilitated nucleocytoplasmic shuttling of the ran binding protein RanBP1 2000

Plafker, K. and Macara, I. G.

Notes: Rabbit Reticulocyte Lysate was used as a source of soluble transport factors to study nuclear localization of a GST-RanBP1 protein into permeabilized cells. Briefly, cells competent for nuclear transport were permeabilized using digitonin. The fusion protein was incubated with the cells in the presence of RRL and an ATP/GTP energy regenerating system. After incubation with the cells, the fusion protein was visualized using immunofluorescence with anti-GST antibodies. (2144)

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J. Gen. Virol. 81, 1103–1109. Mutational evidence that the VPg is involved in the replication and not the movement of Pea enation mosaic virus-1. 2000

Skaf, J.S., Schultz, M.H., Hirata, H. and de Zoeten, G.A.

Notes: The SV Total RNA Isolation System was used to isolate total RNA from 100mg of pea seedling leaves. The isolated RNA was used for Northern blots and RT-PCR. Full transcripts of the Pea enation mosaic virus-1 and Pea enation mosiac virus-2 were produced with T7 RNA Polymerase in vitro and translated in Rabbit Reticulocyte Lysate. (2174)

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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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Genetics 154, 1485-1495. Regulation of the Yeast INO1 Gene. The products of the ino2, ino4 and opi1 regulatory genes are not required for repression in response to inositol. 2000

Graves, J. A. , Henry, S. A.

Notes: Linearized plasmid DNA templates were used in Riboprobe® in vitro Transcription Systems to produce RNA templates for in vitro translation using Rabbit Reticulocyte Lysate Systems, Nuclease Treated. (1117)

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J. Biol. Chem. 274, 3439-3445. Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain. 1999

Hsu, W. , Zeng, L. , Costantini, F.

Notes: Used Promega Rabbit Reticulocyte Lysate to translate an in vitro transcribed RNA for the catalytic subunit of protein phosphatase 2A with 35S-methionine. The resulting radiolabeled protein was used in a protein:protein interaction study with GST-tagged Axin. The authors used Promega's anti-catalytic subunit of protein phosphatase 2A polyclonal antibody for co-immunoprecipitation (antibody has been discontinued). (1017)

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J. Biol. Chem. 274, 457-463. Topology of Euglena chloroplast protein precursors within endoplasmic reticulum to Golgi to chloroplast transport vesicles. 1999

Sulli, C., Fang, Z., Muchhal, U., Schwartzbach, S. D.

Notes: Rabbit Reticulocyte Lysate and Canine Pancreatic Microsomal Membranes (CMMs) were used to study the stop-transfer sequences of pLHCPII. (0316)

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Proc. Natl. Acad. Sci. USA 95, 7463-7468. A chloroplast processing enzyme functions as the general stromal processing peptidase. 1998

Richter, S. and Lamppa, G.K.

Notes: Chloroplast processing enzyme (CPE), was expressed in E. coli as a biotinylated fusion protein, and biotinylation was demonstrated using Promega's SoftLink™ Soft Release Avidin Resin. The singly-biotinylated protein was immobilized onto Streptavidin MagneSphere® Paramagnetic Particles. The immobilized CPE was incubated with the [35S]Methionine-labeled preribulose-1,5-bisphosphate carboxylase/oxygenase activase (preRBCA) and the processed RBCA protein was detected by autoradiography. To further demonstrate the processing, unlabeled preRBCA was processed with the immobilized CPE and the resulting RBCA was analyzed by N-terminal sequencing. The labeled pRBCA was generated with the Rabbit Reticulocyte Lysate System. (0515)

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Mol. Cell 1, 565-574. A novel human WD protein, h-betaTrCP, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif 1998

Margottin, F., Bour, S.P., Durand, H., Selig, L., Benichou, S., Richard, V., Thomas, D., Strebel, K., Benarous, R.

Notes: The protein Vpu or a mutant Vpu were translated with the Rabbit Reticulocyte Lysate System in the presence of Canine Pancreatic Microsomal Membranes. The h-betaTrCP and a mutant were translated in the absence of the membranes. The h-betaTrCP translation reactions were mixed with the Vpu reactions, incubated then centrifuged. The pellet and supernatants were analyzed for the presence of the h-βTrCP protein. (0731)

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Genetics 148, 1865-1874. A proline-rich region in the Zeste protein essential for transvection and white repression by Zeste. 1998

Rosen, C., Dorsett, D., Jack, J.

Notes: Capped zeste transcripts were translated in vitro with either Rabbit Reticulocyte Lysate, Nuclease Treated, or Wheat Germ Extract in the presence of [35S]methionine. (0454)

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Am. J. Physiol. 274, C1380-C1387. Contractile activity-induced adaptations in the mitochondrial protein import system. 1998

Takahashi, M., Chesley, A., Freyssenet, D., Hood, D.A.

Notes: Radiolabeled proteins were translated in the Rabbit Reticulocyte Lysate (RRL). The resultant proteins were assayed for import into Mitochondria. (0293)

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J. Biol. Chem. 273, 7189-7192. Cotranslational ubiquitination of cystic fibrosis transmembrane conductance regulator in vitro 1998

Sato, S., Ward, C.L., Kopito, R.R.

Notes: A modified CFTR sequence containing an hemagglutin tag in the first extracellular loop of the 12 transmembrane domains was translated in the presence of Canine Microsomal Membranes (CMM) with the Rabbit Reticulocyte Lysate. The cDNA sequence was also modified by replacing the 5' UTR with the IRES sequence from the encephalomyocarditis virus. The protein was expressed with or without the CMMs and could be precipitated with an anti-HA antibody. To demonstrate in vitro ubiquitination, 125I-labeled bovine ubiquitin was added to the lysate and 35S-met was replaced with unlabeled met. A lot of detail is provided for all techniques. (0442)

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J. Biol. Chem. 273, 23629. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). 1998

Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., Endou, H.

Notes: The L-type amino acid transporter 1 (LAT1) cloned in this study from glioma cells was translated in vitro with and without Canine Pancreatic Microsomal Membranes (CMMs) using Rabbit Reticulocyte Lysate (RRL). LAT1 is a 56kDa protein with 12 putative transmembrane domains and could be expressed in RRL with and without CMMs. No change in molecular weight was noted for the protein in either case. Another protein used in this study, the CD98 heavy chain of 65kDa, was translated and glycosylated by RRL and CMMs. The glycosylation was removed with endoH. (0955)

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J. Biol. Chem. 273, 23080. KCR1, a membrane protein that facilitates functional expression of non-inactivating K+ currents associates with rat EAG voltage-dependent K+ channels. 1998

Hoshi, N., Takahashi, H., Shahidullah, M., Yokoyama, S., Higashida, H.

Notes: The KCR1 protein, with its putative 12 transmembrane domains, were translated with Rabbit Reticulocyte Lysate with and without Canine Pancreatic Microsomal Membranes. The translated proteins were also treated with endoglycosidase H as directed in referenced protocols. (1014)

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Genetics 150, 1639-1648. Molecular consequences of Ds insertion into and excision from the helix- loop-helix domain of the maize R gene. 1998

Liu, Y., Wang, L., Kermicle, J.L., Wessler, S.R.

Notes: Poly(A)+ RNA was purified with PolyATtract® mRNA Isolation Systems. Anti-Rabbit IgG AP Conjugate, and BCIP/NBT were used in Western blotting. R transcripts were synthesized from the Lc cDNA in the pGEM-7Z(+) Vector, and were used in in vitro translation by Rabbit Reticulocyte Lysate Systems to be used as a positive control in Western. (0781)

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