Classification and expression analyses of homeobox genes from Dictyostelium discoideum.

Homeobox genes are compared between genomes in an attempt to understand the evolution of animal development. The ability of the protist, Dictyostelium discoideum, to shift between uni- and multicellularity makes this group ideal for studying the genetic changes that may have occurred during this transition. We present here the first genome-wide classification and comparative genomic analysis of the 14 homeobox genes present in D. discoideum. Based on the structural alignment of the homeodomains, they can be broadly divided into TALE and non-TALE classes. When individual homeobox genes were compared with members of known class or family, we could further classify them into 3 groups, namely, TALE, OTHER and NOVEL classes, but no HOX family was found. The 5 members of TALE class could be further divided into PBX, PKNOX, IRX and CUP families; 4 homeobox genes classified as NOVEL did not show any similarity to any known homeobox genes; while the remaining 5 were classified as OTHERS as they did show certain degree of similarity to few known homeobox genes. No unique RNA expression pattern during development of D. discoideum emerged for members of an individual group. Putative promoter analysis revealed binding sites for few homeobox transcription factors among many probable factors.

Analysis of rapamycin induced autophagy in Dictyostelium discoideum.

Natural autophagy and autophagic cell death is being studied in the model system, D. discoideum, which has well known genetic and experimental advantages over the other known systems. There is no apoptotic machinery present in this organism which could interfere with the non-apoptotic cell death. The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway which when inhibited by the drug rapamycin induces autophagy. Rapamycin was originally discovered as an anti-fungal agent but its use was abandoned when it was discovered to have potent immunosuppressive and anti-proliferative properties. It is a known drug used today for various cancer treatments and also for increasing longevity in many model organisms. It has a wide usage but its effects on other pathways or molecules are not known. This model system was used to study the action of rapamycin on autophagy induction. Using the GFP-Atg8, an autophagosome marker, it was shown that rapamycin treatment can induce autophagy by an accumulation of reactive oxygen species and intracellular free calcium. Rapamycin suppresses proliferation by induction of cell cycle arrest in the G1 phase. Taken together, the results suggest that the core machinery for autophagy is conserved in D. discoideum and it can serve as a good model system to delineate the action of rapamycin induced autophagy.

Peptide: N- glycanase is expressed in prestalk cells and plays a role in the differentiation of prespore cells during development of Dictyostelium discoideum.

Peptide: N- glycanase (PNGase) enzyme is found throughout eukaryotes and plays an important role in the misfolded glycoprotein degradation pathway. This communication reports the expression patterns of the pngase transcript (as studied by the analysis of β- galactosidase reporter driven by the putative pngase promoter) and protein (as studied by the analysis of β- galactosidase reporter expressed under the putative pngase promoter as a fusion with the pngase ORF) during development and further elucidated the developmental defects of the cells lacking PNGase (png-). The results show that the DdPNGase is an essential protein expressed throughout development and β- galactosidase activity was present in the anterior part of the slug. In structures derived from a null mutant for pngase, the prestalk A and AO patterning was expanded and covered a large section of the prespore region of the slugs. When developed as chimeras with wild type, the png- cells preferentially populate the prestalk/stalk region. When the mutants were mixed in higher ratios, they also tend to form the prespore/spore cells. The results emphasize that the DdPNGase has an essential role during development and the mutants have defects in a system that changes the physiological dynamics in the prespore cells. DdPNGase play a role in development both during aggregation and in the differentiation of prespore cells.

Analysis of Rheb in the cellular slime mold Dictyostelium discoideum: Cellular localization, spatial expression and overexpression.

Dictyostelium discoideum encodes a single Rheb protein showing sequence similarity to human homologues of Rheb. The DdRheb protein shares 52% identity and 100% similarity with the human Rheb1 protein. Fluorescence of Rheb yellow fluorescent protein fusion was detected in the D. discoideum cytoplasm. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that rheb is expressed at all stages of development and in prestalk cells in the multicellular structures developed. When the expression of rheb as a fusion with lacZ was driven under its own promoter, the β-galactosidase activity was seen in the prestalk cells. D. discoideum overexpressing Rheb shows an increase in the size of the cell. Treatment of the overexpressing Rheb cells with rapamycin confirms its involvement in the TOR signalling pathway.

The determination of spatial pattern in Dictyostelium discoideum.

Free-living amoebae of the cellular slime mould Dictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case of Dictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) 'coin tossing', whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) 'chemical kinetics', whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other; and (iii) 'positional information', in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arises in situ.