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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: These authors created a construct encoding a Renilla and firefly luciferase fusion protein to examine the efficiency of site-specific DNA repair. The Renilla gene was taken from the pRL-null Vector and the firefly luciferase gene originated from the pSP-luc+NF Fusion Vector. The fusion construct was created by ligating the C-terminus of the Renilla luciferase gene to the N-terminus of the firefly gene.  The fused proteins were expressed at a constant ratio when transfected into mammalian cells.  Firefly luciferase expression was eliminated by deleting a T at position 213 creating an ochre translational stop codon and placing the downstream sequence out of frame. The mutant protein was then tested for repair using two methods: small fragment homologous replacement and oligonucleotide-mediated repair. The Renilla:firefly expression ratio was tested in several human, murine and simian cell lines and assayed 48 hours post-transfection using the Dual-Luciferase^® Reporter Assay System.  In addition, the repaired plasmids were recovered to verify the sequence correction. (3064)

Notes: These researchers used luciferase-containing T-DNA insertions in Arabidopsis thaliana for gene trapping. Luciferase was chosen because its transient expresion allowed temporal expression studies. Several insertion vectors were constructed and found to have different insertion frequencies.  Vectors containing the luc+  gene had substantially higher insertion rates than native luciferase vectors. Luciferase activity was measured in vivo with a CCD camera or, for longer term studies, with an automated scintillation counter sampling every 15-25 minutes over one week. The application of IRES sites in gene trapping experiments was also investigated using firefly and Renilla luciferases. The Dual-Luciferase^® Reporter Assay System was used to monitor luciferase activity in vitro.  Finally, to sequence the T-DNA insertion sites, genomic DNA was isolated from T₂ seedlings using the Wizard^® Magnetic 96 DNA Plant System and was subsequently amplified and sequenced.  (2787)

Notes: Researchers cloned the luc+ gene from pSP-luc+ into a shuttle vector that could propagate in both Escherichia coli and Streptococcus mutans. These constructs were used to study expression of luciferase in S. mutans. It was found that the luciferase protein has a 95-minute half-life in S. mutans. (3182)

Notes: Luciferase reporter constructs containing either the luc or luc+ gene (from pSP-luc+NF Fusion Vector) were used in in vitro transcription to produce capped mRNA. Luciferase was produced by in vitro translation using either neurospora extract or Promega's Rabbit Reticulocyte Lysate, Nuclease Treated and detected using the Luciferase Assay Reagent. (0208)

Notes: The pSP-luc+NF Fusion Vector was used to produce a luc+ reporter vector for use in the gram negative prokaryote, Azotobacter vinelandii. The Primer Extension System-AMV Reverse Transcriptase was used to find the transcription start site of the bacterial promoter for NAPDH-ferredoxin reductase. (0103)