Notes: This paper describes a potential "specificity checkpoint" for nucleotide incorporation by DNA polymerases. Using Tgo DNA polymerase from Thermococcus gorgonarius as a model system, the authors identified a single key residue (E664) that influenced the ability to incorporate rNTPs. Mutation of E664 to lysine (K) enabled RNA polymerization in the context of another mutation (the steric-gate mutation (Y409G)). The paper describes characterization of the mutant phenotype. As part of the study, luciferase mRNA was generated from the luciferase control DNA using the mutant polymerase. Synthesis and in vitro translation of the synthesized RNA was also evaluated using ssDNA templates, New England Biolabs PURExpress™ protein synthesis kit, and the FluoroTect™ Green_Lys in vitro translation labeling system. (4248)

Notes: Specific neuronal connectivity is thought to be based on the expression of the protocadherins (Pcdh)--a family of membrane adhesion proteins. Each neuron expresses only a specific subset of the Pcdh genes. The authors of this paper used a bioinformatics approach to identify conserved, gene-specific regions upstream of the Pcdh genes. They showed that this specific sequence element (SSE) is involved in transcription regulation together with a conserved sequence element (CSE), and identified a potential interacting protein partner. To demonstrate promoter activity, the SSE-CSE region was cloned upstream of the luciferase gene in the pGL3 Basic Vector and its effect on luciferase expression was evaluated. The authors then isolated protein complexes that bound the SSE-CSE region and characterized the interacting proteins by mass spectrometry. The CCTC binding-factor (CTCF) was identified as a key molecule that binds and activates Pcdh promoters. As part of the study, human CTCF  and the CTCF-binding domain were expressed in the TNT® T7 Quick Coupled Transcription/Translation System. The in vitro expressed proteins were fluorescently labeled using the FluoroTect™ Green_Lys System and were used in EMSA to confirm interaction with the SSE-CSE region. (4249)

Notes: This study characterized interactions between the proton-pumping membrane complex V-ATPase, the Arf nucleotide binding site opener ARNO, and aldolase. The authors used a combination of protein-protein interaction techniques to identify downstream effectors of ARNO and V-ATPase signaling, and identified aldolase as a specific interaction partner of ARNO that could be involved in intracellular trafficking and cytoskeletal modulation. As part of the study, the FluoroTect™ Green_Lys in vitro Translation Labeling System was used to fluorescently label recombinant proteins during in vitro translation reactions. The labeled proteins were used in pull-down experiments. (4250)

Notes: These authors characterized inflammatory caspase substrates using an enzymatic enrichment method for caspase 1,-4 and -5 cleaved proteins and mass-spectrometry. To confirm that caspase-1 cleaved the putative substrates, some substrates were expressed and fluorescently labeled using the TNT® T7 Transcription/Translation System and the FluorTect™ Green_Lys System. The proteins were then treated with recombinant caspase-1, and the progression of the reaction tracked via SDS-PAGE. The authors also used the CytoTox™ ONE Homogeneous Membrane Integrity Assay to track membrane permeabilization in relation to caspase cleavage and IL-1B release. (4252)

Notes: These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag^® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)³⁵S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag^® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use. (3999)

Notes: The Plautia stali virus contains two open reading frames and includes a 5´ internal ribosome entry site (IRES) and an intergenic IRES region. These authors showed that the 5´ IRES was functional and initiated translation in insect cell lysate, but not in rabbit reticulocyte lysate or wheat germ extract. The efficiency of translation mediated by the 5´ IRES region was tested with and without cap analogue using various firefly and Renilla luciferase reporter constructs. They also used deletion mutants to identify the specific regions required for translation initiation. (3942)

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™ Green_Lys 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)

Notes: To prepare expression-ready cDNA clones of 1589 putative full-length ORFs (from human and mouse genes) with an average size of 2.8 kb, a linear trap vector was created. Generated by PCR, this linear plasmid contained gene-specific sequences to allow homologous recombination. In addition, 5–10µg of a plasmid containing the same gene-specific sequence and the linear trap vector were purified using the Wizard^® SV 96 PCR Clean-Up System. The purified DNA was resuspended in water and transformed into E. coli cells. The plasmid purified after the recombination was transferred to a Gateway expression vector and 100–200ng expressed in the TNT^® T7 Quick Coupled Reticulocyte Lysate System with 0.2µl of FluoroTect™ Green_Lys tRNA. The proteins expressed were resolved using SDS-PAGE. (3439)

Notes: In this study, the authors investigated the interaction between the orphan receptors Dax-1 and Ad4bp/SF-1. Specific interaction was detected when the two proteins were used in both yeast and mammalian two-hybrid systems. To further characterize this interaction, an in vitro pull-down assay was performed. Dax-1 protein or a dax-1 mutant lacking an LXXLL motif were transcribed and translated in vitro using the TNT^® T7 Quick System and Fluorotect™ Green_Lys BODIPY labeled tRNA.  The fluorescently labeled protein interacted with a Ad4BP/SF-1-maltose binding protein (MBP) fusion protein immobilized to an amylose resin.  After elution and SDS-PAGE, the fluorescently labeled protein was detected using a Hitachi FMBIO^® II Fluorescence Imaging System. The interaction between Dax-1 and Ad4BP/SF-1 was dependent on the LXXLL motif. (3237)

Notes: Template DNAs encoding c-Fos (118–211 amino acids) and c-Jun (216–318 amino acids) were generated by PCR, purified and then in vitro transcribed using the RiboMAX™ Large Scale RNA Production System.  Following transcription, the RNA was purified and then translated in a wheat germ extract system supplemented with FluoroTect Green_Lys tRNA. The fluorescently labeled proteins generated by the translation reaction were run on a 16.5% Tricine-SDS–PAGE and analyzed with a Molecular Imager FX. Labeling efficiency was calculated by measuring total protein via T7-antibody and determining the amount of fluorescently-labeled protein by fluorescence correlation spectroscopy (FCS). (3084)

Notes: To understand if protein translation occurs in the nucleus of eukaryotic cells, the researchers in this study labeled proteins in vivo by bathing permeablized cells in a translation mix containing radiochemical, biotin or fluorescently tagged amino acids. The study found that all three types of label gave similar results. To fluorescently tag proteins, cells were grown, then permeablized, and bathed in a mix of buffer, creatine phosphokinase, phosphocreatine, GTP, tRNA, aminoacyl-tRNA synthetase, MgCl₂, protease inhibitors, a 1:10 dilution of Fluorotect Green_lys BODIPY labeled lysine-tRNA complex and 50μM amino acid mixture minus lysine. The study revealed that, contrary to previous beliefs, some proteins are transcribed and translated within the nucleus in a coupled manner.  (2744)