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)

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 prokaryotes 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)

Notes: To assay for the effect of experimental compounds on bacterial translation, the test compound was added to the control reaction of pBESTluc™ Control DNA and an E. coli S30 Extract System. It is not specified whether the circular or linear extract system is used. The inhibition was measured as a function of luciferase activity. (0864)

Notes: A his-tagged prothermolysin with Glu-143 of mature enzyme mutated to alanine was expressed in vitro with the E.coli T7 S30 Extract System for Circular DNA. The ³H-leucine labeled protein was used as a substrate for activation. The mutated protein could not be activated. (0732)

Notes: A β-lactamase, PSE-4, was randomly mutated between amino acids 161 to 179. The plasmids containing these mutants were first translated in vitro with the E. coli S30 Extract System for Circular DNA to confirm that expression could occur. The expressed proteins were analyzed by both autoradiography and immunoblotting. The immunoblots were developed with the ProtoBlot^® Western Blot System. (0259)