ABSTRACT
We cloned a protein kinase (DdKinY) from Dictyostelium discoideum by low stringency hybridization using the catalytic domain from DdKinX [B.W. Wetterauer et al., Biochim. Biophys. Acta 1265 (1995) 97-101] as a probe. Both kinases have low sequence similarity to other protein kinases in the databases. However, phylogenetic analysis showed that both kinases cluster with vertebrate LIM kinases due to homology within the catalytic domain.
Subject(s)
Dictyostelium/enzymology , Protein Kinases/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Protozoan , Dictyostelium/growth & development , Gene Expression Regulation, Developmental , Molecular Sequence Data , Protein Kinases/genetics , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Dictyostelium/growth & development , Dictyostelium/genetics , Folic Acid/pharmacology , Lectins , 3',5'-Cyclic-AMP Phosphodiesterases/biosynthesis , 3',5'-Cyclic-AMP Phosphodiesterases/genetics , Animals , Dictyostelium/drug effects , Discoidins , Folic Acid/metabolism , Gene Expression Regulation, Developmental/drug effects , Mutation , PTB-Associated Splicing Factor , Phenotype , Promoter Regions, Genetic , Protozoan Proteins/biosynthesis , Protozoan Proteins/genetics , RNA-Binding Proteins/genetics , Receptors, Cyclic AMP/biosynthesis , Receptors, Cyclic AMP/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Transcription, GeneticABSTRACT
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.
Subject(s)
Dictyostelium/metabolism , Fungal Proteins/biosynthesis , Ammonia/pharmacology , Animals , Cell Count , Culture Media, Conditioned , Cyclic AMP/metabolism , Cyclic AMP/pharmacology , Dictyostelium/cytology , Dictyostelium/genetics , Folic Acid/pharmacology , Fungal Proteins/genetics , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sensitivity and SpecificityABSTRACT
DdKinX codes for 1093 amino acids which are organized in four regions: the N-terminal catalytic domain, a region containing 30% acidic amino acids, tandem repeats of the motif VKVEEPVEE and the C-terminus. Identity with other protein kinases is 25 to 30%. Descendent trees show that DdKinX does not belong to any of the known kinase branches.
Subject(s)
Dictyostelium/enzymology , Protein Kinases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Molecular Sequence Data , Protein Kinases/chemistry , Protein Kinases/isolation & purification , Sequence AlignmentABSTRACT
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.
