Absence of complementation in non-allelic mutants of Dictyostelium discoideum with defects in the transition from growth to development.

In Dictyostelium discoideum, growth and development are mutually exclusive and the transition between the two phases of the life cycle is regulated by the environment. This regulation is disturbed in HBW3, a chemically induced mutant with an unknown molecular defect. The mutant develops rapidly and expresses developmental markers during growth. Here we show that HBW3 fails to complement another mutant which has a similar phenotype: a targeted knock-out of the gdt1 gene. We further show that both mutations are recessive and that both are located on chromosome III, suggesting that the two mutations might be allelic. Molecular analysis, however, demonstrates that the gdt1 gene is not mutated in HBW3. Thus, although a wild-type copy of each gene is present in diploid cell lines, the defects due to the recessive mutations synergize to produce a detectable phenotype. The phenotypic similarities and differences between the two mutants are discussed.

Efficient transformation of Dictyostelium discoideum with a particle inflow gun.

We report experiments to transform Dictyostelium discoideum using a simple home-made particle gun. Stable transformants were obtained at frequencies of up to 2500 clones/microg DNA. This is five times more than we achieve with the same vector using electroporation protocols. We also show that the particle inflow gun can be used for analysis of developmentally regulated gene expression in a transient assay.

Aberrant folate response and premature development in a mutant of Dictyostelium discoideum.

Growth and development are mutually exclusive in Dictyostelium discoideum. The transition between the two stages of the life cycle is regulated by the relative abundance of nutrients and proteins secreted by the cells which reflect population density. At the transition from growth to development, the discoidin genes--developmental markers--are induced by the "quorum" protein PSF. The effect of PSF is counteracted by food bacteria and by folate [8]. We show that folate treatment during growth delays morphologic development. Furthermore, we demonstrate that in a mutant of Dictyostelium discoideum (V188, renamed HBW3), which expresses discoidinI during growth and which develops rapidly [46], discoidinI expression is less sensitive to folate than in wild type cells. Finally, we present evidence that fragments of the discoidinI gamma promoter which are unresponsive to PSF and CM are sufficient for misregulation in the mutant. The only known regulator of these promoter elements is folate. Changes in the expression of other early developmental genes are also shown. Taken together, these data suggest that the reduced sensitivity to folate might be the cause for the "rapid development" phenotype of the mutant and that folate regulates developmental timing.

Cell-density-dependent repression of discoidin in Dictyostelium discoideum.

When Dictyostelium discoideum cells are grown on bacteria, their natural food source, the discoidin genes are induced by cell-density-sensing factors before the food supply is exhausted [11, 18], and expression increases continuously thereafter. This regulation pattern is changed when cells are grown in axenic medium: the discoidins are induced at a considerably lower cell density and are no longer expressed in stationary phase [13]. We have investigated this phenomenon further and show that repression begins when cells are still in exponential growth. It occurs at the level of transcription and involves an element of the discoidin I gamma promoter for which no function has previously been described. Since the effect of high cell density can be mimicked by conditioned medium, it appears that the repression is due to an extracellular signal. This signal is neither ammonia, nor folate, nor cAMP, the known repressors of discoidin expression.

Use of a transactive regulatory mutant of Dictyostelium discoideum in a eucaryotic expression system.

The discoidin proteins of Dictyostelium discoideum are highly expressed during development. The Disc I gamma promoter allows the regulation of heterologous protein expression by experimental conditions. We report conditions under which the promoter activity is efficiently repressed during growth in the wildtype strain AX2. In addition we show that a mutant which overexpresses the discoidins also overexpresses the reporter genes beta-galactosidase, luciferase and CAT 10- to 100-fold when these are placed under the control of a Disc I gamma promoter. This system may be generally useful for the overexpression of genes in Dictyostelium, both for functional studies in vivo and for the production of heterologous proteins for purification.