A NOVEL POSITIVE SELECTION FOR IDENTIFYING COLD-SENSITIVE MYOSIN-II MUTANTS IN DICTYOSTELIUM

We developed a positive selection for myosin heavy chain mutants in Di ctyostelium. This selection is based on the fact that brief exposure t o azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin functi on on a time scale of minutes and has therefore allowed us to select r apid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining w hich mutations lie in sequences of the myosin gene that encode the hea d (motor) domain and localized 27 of 34 mutants to this domain. We rec overed the appropriate sequences from five of the mutants and demonstr ated that they retain their cold-sensitive properties when expressed f rom extrachromosomal plasmids.