By overexpression of modified Escherichia coli 23S rRNAs from multicopy plasmids, ribosomes were prepared that contained mutations in two regions (2447-2450 and 2457-2462) of 23S rRNA. Following mutagenesis and selection, two clones with mutations in the 2447-2450 region (peptidyltransferase center) and six with mutations in the 2457-2462 region (helix 89) were characterized. The mutations were...

The aim of the present study was to evaluate the efficacy of novel nonviral gene delivery systems in cells of musculoskeletal origin. Primary cultures of lapine skeletal muscle cells, lapine articular chondrocytes, human cells from fibrous dysplasia and cell lines established from human osteosarcoma (SAOS-2), chondrosarcoma (CS-1), murine skeletal myoblasts (L8) and fibroblasts (NIH 3T3) were t...

A rapid, continuous method for noninvasively monitoring the effectiveness of several antibacterial agents in real time by using a model of wound infection was developed. This study was divided into three steps: (i) construction of a plasmid to transform Escherichia coli into a bioluminescent variant, (ii) study of the bioluminescent E. coli in vitro as a function of temperature and pH, and (iii...

Understanding luciferase enzymology and the structure of compounds that modulate luciferase activity can be used to improve the design of luminescence-based assays. This review provides an overview of these popular reporters with an emphasis on the commonly used firefly luciferase from Photinus pyralis (FLuc). Large-scale chemical profile studies have identified a variety of scaffolds that inhi...

Although several enzymes known to reside in peroxisomes have been studied extensively, no cis-acting amino acid sequences involved in the transport of these proteins to peroxisomes have been described. As a first step towards the determination of a putative peroxisomal targeting sequence, we have expressed the cDNA encoding the firefly luciferase [Photinus-luciferin:oxygen 4-oxidoreductase (dec...

The selection of a genetic reporter can be difficult because of the wide range of genes available. In order to reduce the selection, we compared the performance of different reporter genes: firefly luciferase (Photinus pyralis lucFF), bacterial luciferase operon (Photorhabdus luminescens luxCDABE), green fluorescent protein (Aequorea victoria gfp), and red fluorescent protein (Discosoma sp. dsr...

We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible for this enhanced thermostability. Replaceme...

We have developed an efficient and reproducible method for RNA transfection, using a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), incorporated into a liposome (lipofectin). Transfection of 10 ng to 5 micrograms of Photinus pyralis luciferase mRNA synthesized in vitro into NIH 3T3 mouse cells yields a linear response of luciferase activity. The...