Complementation ofnpfmutations in diploid amoebae ofPhysarum polycephalum: the basis for a general method of complementation analysis at the amoebal stage

Abstract

Haploid amoebae of Physarum polycephalum may form plasmodia by crossing, a sexual process that involves cellular and nuclear fusions, or by selfing, an asexual process in which the development of a single amoeba into a plasmodium may involve neither cellular nor nuclear fusion. Mutant strains (npf) in which selfing is suppressed were previously assigned to several functional groups on the basis of their ability to cross with one another in certain combinations. In the present study hybrid, diploid amoebae were isolated from both crossing-compatible and incompatible mixtures of npf mutants. The diploid amoebae from mixtures of compatible strains readily formed plasmodia by selfing, but selfing was suppressed in the diploids from incompatible mixtures. Thus the crossing tests between npf mutants may be viewed as complementation tests: their results reflect the differing selfing abilities of the hybrid, diploid amoebae that formed in each mixture. Genetical and environmental factors affecting the efficiency of formation of diploid amoebae were studied, and the diploids were shown to be stable during repeated subcultures. Although diploid amoebae carrying complementing wp/mutations readily formed plasmodia by selfing at 26 °C, they could be cultured without plasmodium formation at 30 °C, a temperature that also inhibited selfing of the haploid npf* strains. Ways are discussed of exploiting this combination of properties in a general procedure for isolating and testing diploids for dominance and complementation of amoebal mutations in P. polycephalum.

Growth, Development and Genetic Characteristics of Physarum polycephalum Amoebae Able to Grow in Liquid, Axenic Medium

“…Tests of diploid amoebae heterozygous and homozygous for the genes controlling growth in axenic medium Three strains of diploid amoebae (RA681-683), assumed to be heterozygous for axe alleles, were isolated from mixtures of amoebae of strains LU352 and OX1 12, a strain unable to grow in axenic medium, by the method described by Anderson & Youngman (1985). Cultures of diploid amoebae assumed to be homozygous for axe alleles were isolated from LU352 and OX112 cultures treated with isopropyl N-(3-~hlorophenyl)carbamate (CIPC) by the procedures described by Dee & Anderson (1984).…”

Cellular events during sexual development from amoeba to plasmodium in the slime mould Physarum polycephalum

“…The isolation of diploid amoebae from mixtures of strains homoallelic for matA was first described by Youngman et al (1 98 1) ; such amoebae have been useful in genetic analysis by allowing dominance and complementation tests on mutants isolated in amoebae (Anderson & Youngman, 1985;Dee et al, 1989). Holt & Huttermann (1 979) observed that nuclear fusion occurred at mitosis in binucleate fusion cells homoallelic for matA, and that cytokinesis gave rise to pairs of uninucleate, presumably diploid daughters.…”

Regulation of Development by the matA Complex Locus in Physarum polycephalum

“…Putative zygotes were isolated essentially as described by Anderson & Youngman (1985) for the isolation of selfing diploid amoebae. Progeny of plasmodia derived from the putative zygotes were classified as follows.…”

“…During sexual development (crossing) pairs of haploid amoebae fuse to form diploid zygotes, which then grow and develop into diploid plasmodia by going through successive mitotic nuclear division cycles in the absence of cell division. Two of the incompatibility loci, matB and matC, act by influencing the probability that pairs of amoebae will fuse (Shinnick et al, 1978;Youngman et al, 1981 ;Anderson & Youngman, 1985). The third locus, matA, seems to have no influence on the probability of amoebal fusion, but affects the events that follow.…”