Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques |
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Authors: | M Neumaier A Braun C Wagener |
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Affiliation: | Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA. |
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Abstract: | Random mutations were generated in the G-protein-coupled receptor (Ste2p) for the tridecapeptide pheromone (alpha-factor) of Saccharomyces cerevisiae. These mutants were screened for variants that responded to antagonists. Because multiple mutations were detected in each mutant receptor recovered from the screen, site-directed mutagenesis was used to create single-site mutant receptors. Three receptors containing mutations F55V, S219P, and S259P were analyzed for their biological responses to various alpha-factor analogs and for their ligand binding profiles. Cells expressing each of the mutant receptors responded to alpha-factor as well as or better than wild-type cells in a growth arrest assay. In contrast, the binding of alpha-factor to the F55V and S219P mutant receptors was at least 10-fold reduced in comparison to wild-type receptor indicating a complex non-linear correlation between binding affinity and biological activity. Cells expressing mutant receptors responded to some normally inactive analogs in biological assays, despite the fact that these analogs had a low affinity for Ste2p. The analysis of these mutant receptors confirms previous findings that the first and sixth transmembrane regions of Ste2p are important for ligand interaction, ligand specificity, and/or receptor activation to initiate the signal transduction pathway. Changes in binding affinity of pheromone analogs to wild-type and mutant receptors indicate that residue 55 of Ste2p is involved with both ligand binding and signal transduction. |
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