Glycogen synthase kinase 3 promotes multicellular development over unicellular encystation in encysting Dictyostelia.

Background: Glycogen synthase kinase 3 (GSK3) regulates many cell fate decisions in animal development. In multicellular structures of the group 4 dictyostelid , GSK3 promotes spore over stalk-like differentiation. We investigated whether, similar to other sporulation-inducing genes such as cAMP-dependent protein kinase (PKA), this role of GSK3 is derived from an ancestral role in encystation of unicellular amoebas.

Stress response of heavy metal mixture present in wastewater and leachate on heat-shock protein 47-transfected cells.

Heavy metals present in water environment and hazardous sites as single compounds or mixture may drastically affect human health. In the present work, we investigated the risk assessment of wastewater effluents and leachate with a focus on three heavy metals-nickel (Ni), cadmium (Cd), and lead (Pb)-and their combined effect on mammalian cells, using Chinese hamster ovary cells transfected with the heat-shock protein (HSP) 47 promoter. The heavy metal mixture model was designed based on the concentrations of metals in wastewater effluents and leachate sampled in Tunisia.

Activated cAMP receptors switch encystation into sporulation.

Metazoan embryogenesis is controlled by a limited number of signaling modules that are used repetitively at successive developmental stages. The development of social amoebas shows similar reiterated use of cAMP-mediated signaling. In the model Dictyostelium discoideum, secreted cAMP acting on 4 cAMP receptors (cARs1-4) coordinates cell movement during aggregation and fruiting body formation, and induces the expression of aggregation and sporulation genes at consecutive developmental stages.

Antimelanogenesis effect of Tunisian herb Thymelaea hirsuta extract on B16 murine melanoma cells.

Skin pigmentation is the result of melanogenesis that occurs in melanocytes and/or melanoma cells. Although melanogenesis is necessary for the prevention of DNA damage and cancer caused by UV irradiation, excessive accumulation of melanin can also cause melanoma. Thus, we focused on the antimelanogenesis effect of an extract from Thymelaea hirsuta, a Tunisian herb.

Regulation of ammonia homeostasis by the ammonium transporter AmtA in Dictyostelium discoideum.

Ammonia has been shown to function as a morphogen at multiple steps during the development of the cellular slime mold Dictyostelium discoideum; however, it is largely unknown how intracellular ammonia levels are controlled. In the Dictyostelium genome, there are five genes that encode putative ammonium transporters: amtA, amtB, amtC, rhgA, and rhgB. Here, we show that AmtA regulates ammonia homeostasis during growth and development.

The cDNA sequencing project.

The Dictyostelium discoideum cDNA sequencing project started in 1995, preceding the genome sequencing project. Altogether, 14 cDNA libraries, including full-length ones, were constructed from five different stages of growth and asexual and sexual development, from which nearly 100,000 randomly chosen clones were sequenced to yield over 150,000 expressed sequence tags (ESTs). The data have been publicized online to facilitate clone distribution and collaboration using the whole clone set for microarray analyses.

Dictyostelium transcriptional host cell response upon infection with Legionella.

Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. Investigation of a 48 h time course of infection revealed several clusters of co-regulated genes, an enrichment of preferentially up- or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co-incubation with Legionella hackeliae and L.

D-threo-tetrahydrobiopterin is synthesized via 1'-oxo-2'-D-hydroxypropyl-tetrahydropterin in Dictyostelium discoideum Ax2.

The biosynthesis of D-threo-tetrahydrobiopterin (DH4, tetrahydrodictyopterin) in Dictyostelium discoideum Ax2 was investigated through the mutant disrupted in the gene encoding sepiapterin reductase (SR) by insertional inactivation. The mutant cells, being completely devoid of SR protein, showed 18.1% of L-erythro-tetrahydrobiopterin (BH4) and 0.

Control of cell type proportioning in Dictyostelium discoideum by differentiation-inducing factor as determined by in situ hybridization.

We have determined the proportions of the prespore and prestalk regions in Dictyostelium discoideum slugs by in situ hybridization with a large number of prespore- and prestalk-specific genes. Microarrays were used to discover genes expressed in a cell type-specific manner. Fifty-four prespore-specific genes were verified by in situ hybridization, including 18 that had been previously shown to be cell type specific.

An orderly retreat: Dedifferentiation is a regulated process.

Differentiation is a highly regulated process whereby cells become specialized to perform specific functions and lose the ability to perform others. In contrast, the question of whether dedifferentiation is a genetically determined process, or merely an unregulated loss of the differentiated state, has not been resolved. We show here that dedifferentiation in the social amoeba Dictyostelium discoideum relies on a sequence of events that is independent of the original developmental state and involves the coordinated expression of a specific set of genes.

Analyses of cDNAs from growth and slug stages of Dictyostelium discoideum.

Dictyostelium is a favored model for studying problems in cell and developmental biology. To comprehend the genetic potential and networks that direct growth and multicellular development, we are performing a large-scale analysis of Dictyostelium cDNAs. Here, we newly determine 7720 nucleotide sequences of cDNAs from the multicellular, slug stage (S) and 10 439 from the unicellular, vegetative stage (V).

A novel gene trap method using terminator-REMI and 3' rapid amplification of cDNA ends (RACE) in Dictyostelium.

We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3' proximal region of the trapped gene.

Changing patterns of gene expression in dictyostelium prestalk cell subtypes recognized by in situ hybridization with genes from microarray analyses.

We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes.

A STAT-regulated, stress-induced signalling pathway in Dictyostelium.

The Dictyostelium stalk cell inducer differentiation-inducing factor (DIF) directs tyrosine phosphorylation and nuclear accumulation of the STAT (signal transducer and activator of transcription) protein Dd-STATc. We show that hyperosmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyperosmotic stress is known to elevate intracellular cGMP and cAMP levels, and the membrane-permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, whereas 8-bromo-cAMP is a much less effective inducer.

Syntaxin 7, syntaxin 8, Vti1 and VAMP7 (vesicle-associated membrane protein 7) form an active SNARE complex for early macropinocytic compartment fusion in Dictyostelium discoideum.

The macropinocytic pathway in Dictyostelium discoideum is organized linearly. After actin-driven internalization, fluid material passes sequentially from endosomes to lysosomes, where molecules are degraded and absorbed. Residual material is exocytosed via post-lysosomal compartments.

A putative serpentine receptor gene tasA required for normal morphogenesis of primary stalk and branch structure in Polysphondylium pallidum.

The fruiting body of Polysphondylium pallidum is composed of whorls of branches along the axis of a primary stalk. In the course of fruiting body formation, the interval between neighboring whorls and the number and the spacing of branches in a whorl are highly regulated. In this study, using restriction enzyme mediated integration mutagenesis, we have obtained a mutant (strain M6226) with thicker and aberrant primary stalk.

A transcriptional profile of multicellular development in Dictyostelium discoideum.

A distinct feature of development in the simple eukaryote Dictyostelium discoideum is an aggregative transition from a unicellular to a multicellular phase. Using genome-wide transcriptional analysis we show that this transition is accompanied by a dramatic change in the expression of more than 25% of the genes in the genome. We also show that the transcription patterns of these genes are not sensitive to the strain or the nutritional history, indicating that Dictyostelium development is a robust physiological process that is accompanied by stereotypical transcriptional events.

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors.

We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5'- and 3'-regions of a target gene.

Transcriptional Regulation of Cell-Type-Enriched Genes during Development in Dictyostelium discoideum Analyzed by Nuclear Run-On Assays: (Dictyostelium discoideum/cell differentiation/cell-type-enriched genes/nuclear run-on assay).

We have previously isolated several cell-type-enriched mRNAs of Dictyostelium discoideum. Since the temporal pattern of appearance and cell-type-enrichment of these RNAs were examined only by determining their accumulation, it was unclear whether their accumulation is regulated at the transcription level or the post-transcriptional level. To distinguish between these two possibilities, we examined the temporal and cell-type-enriched transcription of several of these genes by nuclear run-on assay.