Project description:This project investigated the effect of Cln3-deficiency on protein secretion in the social amoeba Dictyostelium discoideum by performing LC-MS/MS on conditioned media harvested from starved WT and cln3- cells.
Project description:spaA is a Cud-type transcription factor that is essential for spore cell differentiation of Dictyostelium discoideum, a social amoeba. ChIP-seq was performed to identify spaA target genes.
Project description:The social amoeba Dictyostelium discoideum is vulnerable to infection by the pathogen that causes Legionnaire's Disease, Legionella pneumophila. Dictystelium cells lacking the dual-specificity phospahatase DupA are at least partially resistant to infection, and strikingly the expression profile of uninfected dupA null cells mirrors the profile of infected wild-type cells to a considerable extent. This suggests that DupA has a key role in regulating host defence systems.
Project description:ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks. Transcriptional phenotyping during development of abc transporter mutants in Dictyostelium discoideum
Project description:The social amoeba Dictyostelium discoideum integrates into a multicellular organism when individual starving cells aggregate and form a mound. The cells then integrate into defined tissues and develop into a fruiting body that consists of a stalk and spores. Aggregation is initially orchestrated by waves of extracellular cyclic adenosine monophosphate (cAMP) and previous theory suggested that cAMP and other field-wide diffusible signals mediate tissue integration and terminal differentiation as well. Cooperation between cells depends on an allorecognition system comprised of the polymorphic adhesion proteins TgrB1 and TgrC1. Binding between compatible TgrB1 and TgrC1 variants ensures that non-matching cells segregate into distinct aggregates prior to terminal development. Here, we have embedded a small number of cells with incompatible allotypes within fields of developing cells with compatible allotypes. We found that compatibility of the allotype encoded by the tgrB1 and tgrC1 genes is required for tissue integration, as manifested in cell polarization, coordinated movement, and differentiation into prestalk and prespore cells. Our results show that the molecules that mediate allorecognition in D. discoideum also control the integration of individual cells into a unified developing organism and this acts as a gating step for multicellularity. Total 12 samples were used for this analysis. 3 developmental timepoints were analyzed with 2 biological replication. And same 3 developmental timepoint of references (5 RNA mix) with 2 biological replication.
Project description:Pseudomonas aeruginosa is an opportunistic human pathogen, infecting immuno-compromised patients and causing persistent respiratory infections in people affected from cystic fibrosis. Pseudomonas strain Pseudomonas aeruginosa PA14 shows higher virulence than Pseudomonas aeruginosa PAO1 in a wide range of hosts including insects, nematodes and plants but the precise cause of this difference is not fully understood. Little is known about the host response upon infection with Pseudomonas and whether or not transcription is being affected as a host defense mechanism or altered in the benefit of the pathogen. In this context the social amoeba Dictyostelium discoideum has been described as a suitable host to study virulence of Pseudomonas and other opportunistic pathogens.
Project description:Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18-26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase. Keywords: cDNA library; small RNA sequencing Overall design: The aim of this study was to investigate the small RNA (18-26 nt) profile of Dictyostelium discoideum during growth and development. For this reason, we cloned and sequenced pooled small RNAs from growing single cells and from two different multicellular stages (16 and 24 hours of development). cDNA libraries of 18-26 nt D. discoideum RNAs were constructed according to two different protocols (Lee, R.C. and Ambros, V. (2001) Science, 294; Lau, N.C. et al (2001) Science, 294). Briefly, total RNA was isolated from growing D. discoideum AX4 strain cells as well as from cells developed for 16 hours and 24 hours, and the fractions were subsequently pooled. After size fractionation and ligation of a 3’ linker, the RNA was divided into two fractions, one of which was directly ligated to a 5’ linker, thus selecting for small RNAs with 5’ monophosphates. Following RT-PCR, the PCR fragments were cloned and sequenced. The second fraction was subjected to reverse transcription directly after 3’ ligation. Apart from synthesizing a complementary DNA strand, the reverse transcriptase adds a few non-templated C residues at the 3’ end. These C:s were then hybridized to a DNA oligo with three 3’ G:s followed by RT-PCR, cloning and sequencing. This approach is insensitive of the nature of the 5’ end of the small RNA.