Notes: A point mutation in the tyrosine hydroxylase (hTH) enzyme is responsible for a form of dystonia. Mutant and wildtype hTH were expressed by the TNT^® Coupled Reticulocyte Lysate System. Characterization of the mutant enzyme reveals a reduction in activity to approximately 15% of wildtype. (0910)

Notes: Topoisomerase I, Dr1 (a TBP repressor) and various E4BP4 deletion mutant proteins were generated in the TNT^® Coupled Reticulocyte Lysate System using promoters SP6, T3 or T7. The proteins were [³⁵S]methionine-labeled and used in GST pull down assays. E4BP4 mutants that are unable to repress transcription are deficient in Dr1 binding. (1791)

Notes: A point mutation in the tyrosine hydroxylase (hTH) enzyme is responsible for a form of dystonia. Mutant and wild type hTH were expressed by the TNT^® Coupled Reticulocyte Lysate System. Characterization of the mutant enzyme reveals a reduction in activity to approximately 15% of wildtype. (0750)

Notes: The Luciferase T7 Control DNA from the TNT^® Coupled Reticulocyte Lysate System was used to look at cofactors to CD4 necessary for entry of HIV-1. HeLa cells were infected with recombinant vaccinia virus vectors expressing the protein of interest and the T7 RNA Polymerase. Quail QT6 cells were transfected with plasmids encoding CD4, the desired cofactor, and the Luciferase T7 Control DNA. Fusion of the two cells was then quantitated by luciferase activity. (0462)

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. (0860)

Notes: This group evaluated RNA-based screening methods for detection of mutations in hMLH1 and hMLH2. Their results suggest that PTT is useful for preliminary screening tests to localize a mutation, which then can be confirmed by sequencing. The PTT was performed using T7 polymerase and the TNT^® T7 Coupled Reticulocyte Lysate System. (0875)

Notes: The protein truncation tese (PTT) was used to analyze BRCA1 exon 11, which includes ~61% of the coding region of the gene. The observed frequencies of mutations in the BRCA1 gene were not significantly different from expected, based upon phenotype and family history. PTT was performed using the TNT^® T7 Coupled Reticulocyte Lysate System. Each mutation was confirmed by an independent PCR amplification and PTT analysis. (1258)

Notes: [³⁵S]labeled JNK2alpha2 protein was transcribed and translated in vitro using the TNT^® T7 Coupled Reticulocyte Lysate System for use in binding assays with GST-c-Jun, GST-JunB and GST-JunD. The relative binding of JNK2alpha2 to c-Jun was highest, followed by JNK2alpha2 binding to GST-JunB. The binding of JNK2alpha2 to GST-JunD was extremely weak. The results indicate that c-Jun is an excellent substrate for and binds to JNK. Although Jun-B binds JNK, it is not a substrate for JNK. (1794)

Notes: RT-PCR and the Protein Truncation Test (PTT) were used to identify DMD/BMD-associated point mutations that were not detected by DNA-based methods. The RNA method is more sensitive because many of the primers used for methods such as multiplex PCR do not permit the detection of mutations affecting splice sites. The RNA method used in this study screened the total coding region. PTT was performed with the TNT^® Coupled Reticulocyte Lysate System. (0491)

Notes: The TR2 orphan receptor was produced in vitro using the TNT® Coupled Reticulocyte Lysate System. The protein was analyzed by SDS-PAGE and EMSAs. The binding affinity of TR2 for the +55 region of SV40 was then calculated using the ratio between specific DNA-protein binding and free DNA probe. (1841)

Notes: Gag polyproteins were synthesized using the TNT^® Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine. Sedimentation into sucrose gradients and electron microscopy of the lysates were performed to determine if capsid assembly could occur in vitro. The assembly of capsids in this in vitro system does occur and is sensitive to the same mutations that block assembly in vivo. (0430)

Notes: These researchers cloned the FHIT gene by PCR and expressed it in the TNT® Coupled Reticulocyte Lysate System. Synthesized proteins were analyzed using SDS-PAGE and autoradiography. (0596)

Notes: HA and Myc epitope tags were introduced at the C-terminus of Cdc1 and Cdc27 by oligo-directed mutagenesis. Cdc1tag and Cdc27tag were cloned into the pGEM^®-4Z Vector and expressed in the TNT^® T7 Coupled Reticulocyte Lysate System ± [³⁵S]methionine. In GST coimmunoprecipitation assays, Cdc1 interacts with GST-Pol3, and the GST-Pol3 binds preferentially to a truncated Cdc1 translation product. To confirm an interaction observed between Cdc1 and Cdc27, the TNT^®-expressed proteins were mixed together and co-immunoprecipitated using the anti-Cdc27 antibodies Using EGS (ethylene glycol-bis-succinimidylsuccinate) labeled, in vitro translated Cdc1 and Cdc27 were cross-linked. (1771)

Notes: PEA-15 cDNAs were cloned from a mouse astrocytic library, and the clones were linearized and expressed using T3 or T7 Polymerase in the TNT^® Reticulocyte Lysate System either in the presence of [³⁵S]methionine or Transcend™ tRNA. The resulting proteins were analyzed by SDS-PAGE and Western blotting, respectively. A unique 15kDa protein that was recognized by an antibody to PEA-15 was produced in the TNT^® System. (1178)

Notes: Labeled TBP, Rb, E1a, Sp1, TFIIB, TEF-1,SV40 large T antigen (Tag) and BMV were synthesized with [³⁵S]methionine in the TNT^® Coupled Reticulocyte Lysate System. Proteins were assayed for binding to GST-Tag fusion protein. Tag interacts with TBP, TFIIB, Sp1, TEF-1 and the 140kDa subunit of RNA polymerase. Because binding involves the same region of the protein, it probably does not transactivate via concurrent interactions with multiple proteins. (1798)

Notes: [35S]hLEF-1 (human lymphoid enhancer factor-1) protein fragments were generated in the TNT® Coupled Reticulocyte Lysate System. These proteins were incubated with GST-pendulin and GST-mSrp1 bound to glutathione Sepharose beads. Mouse pendulin and mSrp1 bind to the B box in the HMG DNA binding domain of hLEF-1. (1811)

Notes: In this paper, transcription/translation was performed using the TNT^® Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine. Factor VIII fragments were incubated with mAbs and subjected to electrophoresis on 12% denaturing gels. These fragments helped to localize the epitope of the CLB-CagA antibody. (1766)

Notes: [35S]labeled E1 and E2 were expressed in the TNT® Coupled Reticulocyte Lysate System. The proteins were analyzed by SDS-PAGE and then used in DNA-protein immunoprecipitation assays. The E1 and E2 proteins were also tested for interaction in DNA binding assays. The E1 and E2 proteins cooperatively bind the origin, and this event requires E2 binding sites. (1835)

Notes: Transcription/translation of circular template was performed using the TNT^® T7 Coupled Reticulocyte Lysate System. The results helped to characterize the human prostate-restricted transglutaminase. (1201)

Notes: RT-PCR and PTT detected truncated peptides produced from the mRNA transcripts of mutant CFTR genes. Due to the large size of the gene for CFTR, RT-PCR and the PTT are attractive tests for screening for frameshift mutations in the gene. The PTT was performed using an aliquot of nested PCR product (consisting of the CFTR cDNA fused to the T7-promoter) in the TNT® T7 Coupled Reticulocyte Lysate System. (1869)

Notes: The pGEM^®-7zf(-) vector was used for subcloning cDNAs. The pCAT^®3-Control Vector was used as a control in studies of CAT reporter constructs in CV1 and HeLa cells. GST and GST-YY1 fusion proteins were produced using the TNT^® T7 Coupled Reticulocyte Lysate System. (0141)

Notes: XTcf-3, β-catenin and their derivatives were transcribed and translated in the T7 TNT^® Coupled Reticulocyte Lysate System. Gel retardation assays were performed with dsDNA probe from the T cell receptor a enhancer. The β-catenin and DN/C/beta-catenin yielded supershifted bands. Deletion of the first 31 amino acids abrogated the interaction with beta-catenin. (0655)

Notes: Xenopus sonic hedgehog (XHH) cDNA clones were transcribed and translated using the TNT® T7 Coupled Reticulocyte Lysate System. Two smaller variants were found. The smaller proteins may represent proteolytic products. (0338)

Notes: The thyroid hormone receptor-alpha was tested for interaction of its A/B domain with TFIIB. The labeled proteins were prepared using the TNT^® Coupled Reticulocyte Lysate System with L-[³⁵S]cysteine or L-[³⁵S]methionine. The labeled proteins were incubated with GST-TFIIB and shown to interact in vitro. (1796)

Notes: [³⁵S]methionine-labeled HA-Bak and HA-BakDeltaGD were synthesized in vitro using the TNT^® Coupled Reticulocyte Lysate System. These proteins were used in interaction assays with GST-Bcl-xL to detect interactions between regions of Bak and Bcl-xL. (1790)