Expression of starvation-induced genes in zygotes ofDictyostelium discoideum

Abstract

Two cDNA fragments induced in developing zygotes of Dictyostelium discoideum were isolated by mRNA differential display. The relevant genes were also found to be expressed during asexual development, suggesting that sexual and asexual development share common molecular mechanisms in D. discoideum. Key wordr Developmental genes~ Dictyostelium disco/deum~mRNA differential display~Zygote developmentDictyostelium discoideum proliferates as a unicellular ameba, but upon starvation the cells gather and form multicellular structures known as fruiting bodies. Alternatively, they sexually fuse to form zygotic giant cells, which engulf surrounding cells and eventually develop into macrocysts (Nickerson and Raper 1973, O'Day 1979). Simplicity of the sexual development, fidelity of molecular biological techniques, and establishment of synchronous cell fusion system show that it may serve as a useful experimental system for cell recognition, membrane fusion, and zygote development.Molecular analyses of sexual cell fusion in D. discoideum have been performed to a limited extent (Urushihara 1997), but that for zygote development it is very scarce. In the present study we analyzed the pattern of gene expression during zygote development using the mRNA differential display method, and found that two novel genes are induced shortly after cell fusion.Complementary heterothallic strains of D. discoideum, NC4 and HM1, were used. Zygote giant cells were obtained by co-incubating sexually mature cells of NC4 and HM1 on a rotary shaker at 120 rpm for 30 min at 22 C as described previously (Saga eta/. 1983). Where necessary, giant cells thus obtained (crude GCs) were freed of unfused cells by differential centrifugation to yield purified GCs. Both crude and purified GCs developed into macrocysts after a 48 hr incubation on nitrocellulose membrane filters layered on wet support pads.The necessity of de novo mRNA synthesis for macrocyst formation was determined using actinomycin D, an inhibitor Present address: Doi Bioassymmetry Project, ERATO, JST, Tsukuba Research Consortium, Tsukuba, Ibaraki, 300-26 Japan of RNA polymerasell. The presence of 125/zg/ml of actinomycin D for 3 hr of the initial development suppressed macrocyst development (data not shown), suggesting that the expression of new genes occurs during this period induced by cell fusion.Messenger RNA differential display (mRNAdd) was performed by a standard protocol (Liang et a/. 1993) using RNA map kit (GeneHunter; Brooklyn, MA. USA) for total RNA prepared from the cells at 0, 3, and 6 hr of sexual development.From roughly 3,000 cDNA fragments amplified by combinations of 3 anchored oligo (dT) and 20 arbitrary primers, two fragments, designated as G9b and A5, were reproducibly found to be present in the samples of 3 and 6 hr of development but absent in that of 0 hr (Fig. la). These fragments were then extracted from the electrophoresis gel, radiolabeled, and used as probes for northern hybridization to detect expression of the relevant genes (G9b and A5, respectively). They we...