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.     

Dictyostelium discoideum: a model system for differentiation and patterning

In Dictyostelium, development begins with the aggregation of free living amoebae, which soon become organized into a relatively simple organism with a few different cell types. Coordinated cell type differentiation and morphogenesis lead to a final fruiting body that allows the dispersal of spores. The study of these processes is having increasing impact on our understanding of general developmental mechanisms. The availability of biochemical and molecular genetics techniques has allowed the discovery of complex signaling networks which are essential for Dictyostelium development and are also conserved in other organisms. The levels of cAMP (both intracellular and extracellular) play essential roles in every stage of Dictyostelium development, regulating many different signal transduction pathways. Two-component systems, involving histidine kinases and response regulators, have been found to regulate intracellular cAMP levels and PKA during terminal differentiation. The sequence of the Dictyostelium genome is expected to be completed in less than two years. Nevertheless, the available sequences that are already being released, together with the results of expressed sequence tags (ESTs), are providing invaluable tools to identify new and interesting genes for further functional analysis. Global expression studies, using DNA microarrays in synchronous development to study temporal changes in gene expression, are presently being developed. In the near future, the application of this type of technology to the complete set of Dictyostelium genes (approximately 10,000) will facilitate the discovery of the effects of mutation of components of the signaling networks that regulate Dictyostelium development on changes in gene expression.     

Dictyostelium discoideum: a promising centrosome model system

The centrosome of the slime mould Dictyostelium discoideum displays a morphology markedly different from centriolar centrosomes or yeast spindle pole bodies, while fulfilling the same conserved functions in the organization of the microtubule cytoskeleton. Recent advances suggest that the Dictyostelium centrosome may offer an interesting model system, usefully complementing other well-studied centrosome models. The establishment of an isolation procedure and the generation of a range of monoclonal antibodies have been achieved, which are important pre-requisites for biochemical investigation. Furthermore, the role of the centrosome in cell motility and centrosome duplication process have been investigated using cells with GFP-labelled centrosomes.     

Sequence and expression of annexin VII of Dictyostelium discoideum.

Sequence analysis reveals that a gene expressed during growth and early development of Dictyostelium discoideum encodes a polypeptide which exhibits extensive similarity with annexins, a family of calcium/phospholipid binding proteins. Comparison of the amino acid composition of the N-termini suggests that the Dictyostelium annexin is a homologue of human synexin, also referred to as annexin VII.     

Establishment of the nuclear location of Dictyostelium discoideum plasmids

The nuclear location of the Dictyostelium discoideum plasmids was studied using a biochemical approach based on the presence of plasmid sequences in nucleosomes. This analysis revealed that all four of the known plasmids (Ddp1, Ddp2, Ddp3, Ddp5) are present in chromatin. This evidence establishes that the D. discoideum plasmids are not cytoplasmic but located in the nucleus. D. discoideum is unique among eukaryotes in possessing a group of nonhomologous endogenous plasmids in its nucleus. These plasmids are excellent starting material for construction of nuclear transformation and expression vectors. Such vectors upon transformation into D. discoideum are also present in chromatin as expected for DNA located in the nucleus.     

Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum.

An in vitro endosome fusion assay using Dictyostelium discoideum is described. The method requires endocytosis of anti-dinitrophenol (DNP) IgG or DNP-derivitized beta-glucuronidase into two sets of cells. After homogenizing the cells, the vesicles were mixed, and fusion was measured by quantitating immune complex formation between DNP-beta-glucuronidase and anti-DNP IgG. Fusion was dependent upon ATP, temperature, pH, ionic strength, and cytosol and sensitive to detergent, dilution, trypsin, N-ethylmaleimide, and guanosine 5'-3-O-(thio)triphosphate. Although weak bases, ionophores, hadacidin, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and caffeine inhibit endocytosis in vivo, these reagents had no affect on in vitro endosome fusion. Comparison of Dictyostelium with mammalian cells showed differences in the temperature, pH, and salt requirements for fusion, possibly reflecting differences in the life-styles of various cell types. Like mammalian cells, Dictyostelium required GTP-binding protein(s) and an N-ethylmaleimide-sensitive factor for endosome fusion. Thus, the mechanism driving endosome fusion may have been conserved throughout evolution. Electron microscopic studies confirmed in vitro endosome fusion and revealed endosomes were being engulfed by other endosomes, resulting in formation of multivesicular elements (i.e. autophagic vesicles). This system may be useful for characterizing mutations, evolution, and developmental regulation along the endocytic pathway.     

Thymidine-requiring mutants of Dictyostelium discoideum.

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.     

Improvement of a synthetic medium for Dictyostelium discoideum

Dictyostelium discoideum is of considerable interest as an expression system for the production of proteins of high value. The cultivation of this social amoeba is not as easy as that of other common microbial expression systems. Wildtype strains grow on bacteria. Mutant strains growing on axenic media reach cell densities of 1–2×10⁷ ml⁻¹ when cultivated in commonly used complex media. A totally synthetic medium formulated by Franke and Kessin (Proc. Natl. Acad. Sci. USA 74 (1977) 2157) has become popular and allows cell densities of about 3×10⁷ ml⁻¹ to be obtained. This medium (FM) is being improved mainly on the basis of the analysis of limitations with respect to amino acids. With this improved synthetic medium (SIH) cell densities in the order of 5–6×10⁷ ml⁻¹ have been achieved.     

Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system

Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.     

Codon preference in Dictyostelium discoideum.

Dictyostelium discoideum is of increasing interest as a model eukaryotic cell because its many attributes have recently been expanded to include improved genetic and biochemical manipulability. The ability to transform Dictyostelium using drug resistance as a selectable marker (1) and to gene target by high frequency homologous integration (2) makes this organism particularly useful for molecular genetic approaches to cell structure and function. Given this background, it becomes important to analyze the codon preference used in this organism. Dictyostelium displays a strong and unique overall codon preference. This preference varies between different coding regions and even varies between coding regions from the same gene family. The degree of codon preference may be correlated with expression levels but not with the developmental time of expression of the gene product. The strong codon preference can be applied to identify coding regions in Dictyostelium DNA and aid in the design of oligonucleotide probes for cloning Dictyostelium genes.     

Evidence for a Sex Hormone in Dictyostelium discoideum

A dialyzable sex hormone is produced by one strain of Dictyostelium discoideum (NC-4) that induces macrocyst formation (sexual stage) in its opposite mating type (V-12).     

Evidence for a membrane-bound pyrophosphatase in Dictyostelium discoideum

Plasma membrane enriched fractions of Dictyostelium discoideum contain a Des-insensitive ATPase activity that can be fractionated by DEAE-Sephacel into a major vanadate-sensitive activity and a minor vanadate-insensitive activity. The vanadate-insensitive activity hydrolyzed pyrophosphate considerably more rapidly than ATP or any other substrate tested, and the enzyme was therefore designated a pyrophosphatase. The enzyme had no activity on AMP or p-nitrophenyl phosphate. The pyrophosphatase activity was maximal at alkaline pH values and stimulated by Mg2+ but not by Ca2+, properties of the enzyme that are very similar to those of the previously characterized pyrophosphatases of the plant tonoplast membrane. The pyrophosphatase activity of total membrane extracts changed very little during Dictyostelium differentiation.     

Phosphotyrosine-containing proteins in Dictyostelium discoideum.

Phosphotyrosine-containing proteins in Dictyostelium discoideum were detected by immunoblot analysis and immunoprecipitation using a monoclonal anti-phosphotyrosine antibody. The iodinated antibody recognized on bots a cluster of 205-220 kDa polypeptides and bands of 107 and 60 kDa. The 107 and 60 kDa polypeptides and, in addition, a 82 kDa one became phosphorylated on tyrosine when the immunoprecipitate was incubated with [gamma-32P]ATP. In preparations from differentiating cells the intensity of the label was increased in the 60 kDa band and decreased in the 107 and 205-220 kDa bands.     

Evidence for phosphoryl moieties in a proteinase from Dictyostelium discoideum.

A proteinase (called Proteinase I) present in myxamoebae of the cellular slime mold, $\text{Dictyostelium}\text{discoideum}$, was labeled $\text{in}\text{vivo}$ with [³²P] by growth of cells on media containing [³²P] orthophosphate. The labeled proteinase was purified to apparent homogeneity and characterized by dissociation chromatography and quantitative immune-precipitin analysis. Based upon the results of these studies it was concluded that phosphoryl moieties were tightly associated (presumably covalently bonded) with the polypeptide subunits of Proteinase I.     

Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as "slugs." Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.