The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes encode ~12,500 predicted proteins, a high proportion of which have long repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal rDNA element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal/fungal lineage after the plant/animal split, but Dictyostelium appears to have retained more of the diversity of the ancestral genome than either of these two groups.The amoebozoa are a richly diverse group of organisms whose genomes remain largely unexplored. The soil-dwelling social amoeba Dictyostelium discoideum has been actively studied for the past fifty years and has contributed greatly to our understanding of cellular motility, signalling and interaction 1 . For example, studies in Dictyostelium provided the first descriptions of a eukaryotic cell chemo-attractant and a cell-cell adhesion protein 2, 3 .Dictyostelium amoebae inhabit forest soil consuming bacteria and yeast, which they track by chemotaxis. Starvation, however, prompts the solitary cells to aggregate and to develop as a true multicellular organism, producing a fruiting body comprised of a cellular, cellulosic stalk supporting a bolus of spores. Thus, Dictyostelium has evolved mechanisms that direct the differentiation of a homogeneous population of cells into distinct cell types, regulate the proportions between tissues and orchestrate the construction of an effective structure for the dispersal of spores 4 . Many of the genes necessary for these processes in Dictyostelium were Eichinger et al. Page 2 Nature. Author manuscript; available in PMC 2006 January 27. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript also inherited by metazoa and fashioned through evolution for use within many different modes of development.The amoebozoa are also noteworthy as representing one of the earliest branches from the last common ancestor of all eukaryotes. Each of the surviving branches of the crown group of eukaryotes provides an example of the ways in which the ancestral genome has been sculpted and adapted by lineage-specific gene duplication, divergence and deletion. Comparison between representatives of these branches promises to shed light not only on the nature and content of the ancestral eukaryotic genome, but on the diversity of ways in which its components have been adapted to meet the needs of complex organisms. The genome of Dictyosteliu...

Classical epistasis analysis can determine the order of function of genes in pathways using morphological, biochemical and other phenotypes. It requires knowledge of the pathway's phenotypic output and a variety of experimental expertise and so is unsuitable for genome-scale analysis. Here we used microarray profiles of mutants as phenotypes for epistasis analysis. Considering genes that regulate activity of protein kinase A in Dictyostelium, we identified known and unknown epistatic relationships and reconstructed a genetic network with microarray phenotypes alone. This work shows that microarray data can provide a uniform, quantitative tool for large-scale genetic network analysis.

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. A defect in one of these genes, the histidine kinase dhkA, alters the kinetics of dedifferentiation and uncouples the progression of dedifferentiation events. These observations establish dedifferentiation as a genetically determined process and suggest the existence of a developmental checkpoint that ensures a return path to the undifferentiated state. Dedifferentiation is the progression of cells from a more differentiated to a less differentiated state. It is observed in a variety of processes such as cancer, organ regeneration, and stem cell renewal, but it has been difficult to study because there are few experimentally tractable systems that dedifferentiate (1-4). Dedifferentiation in Dictyostelium is an experimentally tractable process. During development, starving cells aggregate and undergo synchronous morphological transitions until they form fruiting bodies after 24 hr (5). If the multicellular structures are disaggregated and incubated in nutrient medium, the cells dedifferentiate. Dedifferentiation is characterized by a loss of developmental markers and a subsequent gain of proliferative capacity (6, 7, 45), but these characteristics do not prove that dedifferentiation is a regulated process. The most convincing evidence in support of regulation has been the observation that a mutant strain (HI4) was defective in dedifferentiation (8,9).An argument against the idea that dedifferentiation is a regulated process comes from the observation that dedifferentiation occurs at different rates, depending on the developmental stage at which the cells were disaggregated (6, 10-13). This dependence may indicate that each developmental stage has a dedicated dedifferentiation program that is executed at a different rate, or that dedifferentiation is a stochastic event whereby cells lose developmental markers and regain growth markers.The purpose of this work was to test whether dedifferentiation is a regulated process by comparing the molecular progression of cells from different developmental stages. We propose that if we found a common set of molecular changes, which is independent of the initial developmental stage, that finding would support the regulated process hypothesis. We used microarray transcriptional profiling to detect changes in the pattern of global gene expression during dedifferentiation. These transcriptional changes reveal physiological changes without prejudice as to what processes are involved (14-18), making them suitable for testing whether...

The tag genes of Dictyostelium are predicted to encode multi-domain proteins consisting of serine protease and ATP-binding cassette transporter domains. We have identified a novel tag gene, tagA, which is involved in cell type differentiation. The tagA mRNA accumulates during the first four hours of development,whereas TagA protein accumulates between two and ten hours of development and decreases thereafter. Wild-type cells express tagA in prespore cells and mature spores, defining tagA expression as prespore specific. However, tagA mutant cells that activate the tagA promoter do not sporulate, but instead form part of the outer basal disc and lower cup of the fruiting body. tagA mutant aggregates elaborate multiple prestalk cell regions during development and produce spores asynchronously and with low viability. tagA mutants produce about twice as many prestalk cells as the wild type as judged by a prestalk cell reporter construct. When mixed with wild-type cells, tagA- cells become overrepresented in the prestalk cell population, suggesting that this phenotype is cell-autonomous. These results suggest that TagA is required for the specification of an initial population of prespore cells in which tagA is expressed. Expression profiling uncovered a delay in the transcriptional program between 2 and 6 hours, coincident with TagA expression, revealing an early function for TagA. TagA also appears to play a general role in cell fate determination since tagA mutants express a spore coat protein gene (cotB) within vacuolated cells that form part of the stalk and they express a prestalk/stalk-specific gene (ecmB)within cells that become spores. The expression of TagA at two hours of development, the observed coincident delay in the transcriptional program and the subsequent mis-expression of cell-type specific genes provide evidence for cell fate determination beginning in some cells much earlier than previously believed.