The isolation and initial characterization of dibucaine-resistant variants of a mouse lymphocytic cell line

Abstract

Mutation in Cultured Mammalian Cells Criteria for the classification of phenotypic variants as mutants 5 Characteristics of mammalian cell lines important for mutant isolation 8 Cell hybridization 10 Induced mutagenesis 10 Selection of membrane, mutants of cultured mammalian cells 15 Information obtained from studies of mutations affecting the plasma membrane Membrane Structure The Fluid Mosaic Model of membrane structure Local Anesthetic Action on Cell Membranes Effects of local anesthetics on cells with excitable membranes Physical-chemical characteristics of local anesthetics 26 Theories of local anesthetic action on excitable cells Cell surface difference and local anesthetic activity Effects of local anesthetics on cells with nonexcitable membranes 41 MATERIALS AND METHODS 55 Cell Lines 55 Media and Chemicals 59 Cloning in Soft Agar Suspension 59 Mutagenesis 60 Selection of Temperature-Sensitive Mutants 60 Direct Selection of Dibucaine-Resistant Mutants at 37°C 62 iii Mutagen-Dose Response Curve 63 Cell Counts 63 Studies to Determine the Toxic Effects of Dibucaine on Cell Growth Polyethylene Glycol-Induced Cell Fusion Cell Hybridization Chromosome Analysis Fluorescein-Concanavalin A Staining Protocol for Fluorescent Microscopy RESULTS GF7 sensitivity to Dibucaine Induction and Isolation of Dibucaine-Resistant Variants Selection of temperature-sensitive mutations to dibucaine resistance 76 Single-step selection in suspension culture at 37°C 83 Expression time 87 Initial Characterization of the Dibucaine-Resistant Phenotype 90 Growth response in the presence of dibucaine 91 Cross resistance to other local anesthetics 93 Effects of ouabain on the growth of dibucaineresistant variants 97 Plasma membrane fusion ability of Dib-5 and Dib-10 99 Redistribution of concanavalin A receptors 103 R Genetic Analysis of Dib Variants 105 Phenotypic stability 105 EMS concentration versus induction of dibucaineresistant variant clones 109 Cell hybridizations 112 iv DISCUSSION 120 Recovery of Dibucaine-Resistant Cells The frequency of occurrence of dibucaine-surviving cells 121