CHARACTERIZATION OF NPF MUTANTS IDENTIFYING DEVELOPMENTAL GENES IN PHYSARUM

In Physarum polycephalum, uninucleate haploid amoebae develop into mac roscopic multinucleate plasmodia. Wild-type, sexual development is tri ggered when two amoebae carrying different alleles of matA fuse to for m a zygote which develops into a diploid plasmodium. Mutations in the matA genetic region give rise to apogamic strains in which a single ha ploid amoeba can develop into a haploid plasmodium. An essential stage in both sexual and apogamic plasmodium formation is an extended cell cycle in uninucleate cells, which ends with the formation of a binucle ate cell by mitosis without cytokinesis. Using a 'brute force' screeni ng method, we have isolated mutants blocked in apogamic plasmodium dev elopment. Genetic analysis showed that the mutations we have identifie d were unlinked to matA, unlike mutations previously identified follow ing an enrichment step. Most of the loci revealed by our screen were r epresented by only one allele, indicating that further screening shoul d lead to the identification of additional genes required for plasmodi um development. Phenotypic analysis showed that different mutants were blocked at different stages of plasmodium formation. Some of the muta tions blocking apogamic development at an early stage, close to the st art of the long cell cycle, failed to block sexual development in zygo tes homozygous for the mutation. Since the two modes of plasmodium for mation differ only in the initiation of development, these mutations p resumably interfere with the initiation process. In the remaining muta nts, in which both sexual and apogamic development were blocked, devel opment first became abnormal towards the end of the long cell cycle. T his suggested that the wildtype gene products were required by this ti me and was consistent with previous evidence that many changes in cell ular organization and gene expression occur during the long cell cycle . Each of these mutants showed a different terminal phenotype and some aspects of plasmodium development occurred normally although others w ere blocked, suggesting that development involves multiple pathways ra ther than a dependent sequence of events. Phenotypic analysis of doubl e mutants supported this conclusion and also revealed epistatic intera ctions, presumably due to blocks in the same pathway. In several of th e mutants, terminally differentiated cells died by an apoptosis-like m echanism; since this was never observed in vegetative cells, it was pr esumably triggered by the failure of development. Phenotypic analyses of additional mutants will extend our understanding of the pathways in volved in plasmodium development.