A novel positive selection for identifying cold-sensitive myosin II mutants in Dictyostelium.
We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids.
D000818:Animals; D001386:Azides; D001483:Base Sequence; D002874:Chromosome Mapping; D003080:Cold Temperature; D004023:Dictyostelium / Q000235:genetics*; D005819:Genetic Markers; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009154:Mutation; D009218:Myosins / Q000235:genetics*; D015345:Oligonucleotide Probes; D010641:Phenotype; D010957:Plasmids; D012641:Selection, Genetic; D019810:Sodium Azide
D001386:Azides; D005819:Genetic Markers; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; D019810:Sodium Azide; D009218:Myosins