Benzimidazoles in Physarum: Differential Expression O F &tubulin Genes

Physarum displays two vegetative cell types, uninucleate myxamoebae and multinucleate plasmodia. Mutant myxamoebae of Physarum resistant to the antitubulin drug methylbenzimidazole-2-yl-carbamate (MBC) were isolated. All mutants tested were cross-resistant to other benzimidazoles but not to cycloheximide or emetine. Genetic analysis showed that mutation to MBC resistance can occur at any one of four unlinked loci, benA, benB, benC or benD. MBC resistance of benB and benD mutants was expressed in plasmodia, but benA and benC mutant plasmodia were MBC sensitive, suggesting that benA and benC encode myxamoeba-specific products. Myxamoebae carrying the recessive benD2lO mutation express a &tubulin with novel electrophoretic mobility, in addition to a &tubulin with wild-type mobility. This and other evidence indicates that benD is a structural gene for &tubulin, and that at least two 8-tubulin genes are expressed in myxamoebae. Comparisons of the p-tubulins of wildtype and benD210 strains by gel electrophoresis revealed that, of the three (or more) &tubulin genes expressed in Physarum, one, benD, is expressed in both myxamoebae and plasmodia, one is expressed specifically in myxamoebae and one is expressed specifically in plasmodia. However, mutation in only one gene, benD, is sufficient to confer MBC resistance on both myxamoebae and plasmodia. OST of the effects of benzimidazole compounds in vivo are consistent M with specific action of the drugs on tubulin and or microtubules. These effects include depolymerization of microtubules in mammalian cells (DEBRABANDER et al. 1976, 1981; HAVERCROFT, QUINLAN and GULL (1981) and mitotic instability and inhibition of mitosis in Aspergillus, yeast and slime molds (HASTIE 1970; DAVIDSE 1973; HAMMERSCHLAC and SISLER 1973; WILLIAMS and BARRAND 1978; CAPPUCCINELLI, F CHETTI and RUBINO 1979; QUINLAN, POCSON and GULL 1980; WELKER and WILLIAMS 1980; WILLIAMS 1980; FANTES 1982; WALKER 1982; WOOD 1982; WOOD and HARTWELL 1982). Several benzimidazoles inhibit the polymerization of purified mammalian brain tubulin into microtubules in vitro (HOEBEKE, VAN NIJEN and DEBRABANDER 1976; FRIEDMAN and PLATZER 1978). HOEBEKE, VAN NIJEN and DEBRABANDER (1976) showed that nocodazole competitively inhibits binding of colchicine to purified brain tubulin, suggesting that the mode of action of ' Permanent address: The Biological Laboratories, University of Kent, Canterbury, CT2 7NJ. England. Genetics 108: 123-141 September, 1984