Dictyostelium Genomics Chapter Abstracts

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The genome of Dictyostelium discoideum was recently sequenced by an International Consortium with participants at Baylor College of Medicine in Texas, the Institute of Molecular Biotechnology in Germany, and the Sanger Centre in England. It was assembled to a high level of accuracy on the basis of classical and physical maps of the 6 chromosomes that had been built up over the years and then refined by HAPPY mapping using PCR primers designed from the raw reads of the consortium. Annotation of the sequence predicted 13,318 genes for proteins of at least 50 amino acids in length. The total number of amino acids encoded (the proteome) is more than double that in yeast and rivals that of metazoans. The genome sequence shows all the proteins available to Dictyostelium as well as definitively showing which domains have been lost or evolved after Dictyostelium diverged from the line leading to metazoans. Genomics opens the door to determining the expression patterns of all the genes during growth and development using microarrays. This approach has already uncovered a wealth of new markers for the stages of development and the various cell types. Transcription factors and their cis-regulatory sites that account for the surprising complexity of Dictyostelium development can be analyzed much more easily now that we have the complete sequence. The pace of progress will undoubtedly increase in the post-genomic era.

Chapter 2
dictyBase: Using the Genome to Organize Dictyostelium Biology
Rex L. Chisholm

The completion of the Dictyostelium genome sequence represents an important milestone that opens the doors for a large variety of functional genomics and genetics studies. The success of these future studies, as well as those for which Dictyostelium has long been a leading experimental system, is greatly facilitated by efficient and user friendly access to the genome sequence and the associated biological knowledge that has been produced by the Dictyostelium community. dictyBase provides a single portal for accessing this data, including chromosome, gene and protein sequence, functional annotations using Gene Ontology terms, relevant phenotypes, expression patterns, curated literature and access to mutants through the Dictyostelium Stock Center. This chapter highlights many of the key features of dictyBase and provides an introduction to using dictyBase to access this information.

Chapter 3
Metabolic Pathways
Samuel H. Payne

Homologs of the enzymes for glucose catabolism as well as in metabolic pathways for amino acid, nucleotide and vitamin biosynthesis were assigned from the proteins predicted from the Dictyostelium genome. Pathways leading to the synthesis of 11 amino acids appeared to be missing one or more critical enzymes. These amino acids are provided for in the defined medium, FM, used for axenic growth. The pathways leading to asparagine, cysteine, glutamate and proline appeared to be intact. Although these amino acids are components of FM, a new minimal medium lacking these 4 amino acids was shown to support growth of Dictyostelium strain AX4. The minimal medium had to include either serine or glycine, which are interconvertable, since the biosynthetic pathway leading to serine appears to have recently been broken. The list of essential amino acids for Dictyostelium is almost identical to that of humans. Dictyostelium appears to have lost the enzymes of the urea cycle although it can still generate urea by conversion of arginine to ornithine. The Dictyostelium genome was also analyzed to find genes shown to be essential for growth of yeast (S. cerevisiae) and embryogenesis of zebra fish (Danio rerio). These essential genes were more highly conserved than expected from comparison of the total complement of genes.

Chapter 4
Multigene Families of Dictyostelium
Christophe Anjard

Multigene families arise from gene duplications followed by divergence or deletion of one or more gene copies. Some families have an ancient origin that predates eukaryotes while others appeared later on. In some cases, recent amplifications have generated new families. Half of the Dictyostelium proteins show significant sequence similarity with other proteins and could be clustered into groups. This approach allowed the entire set of Dictyostelium clusters to be graphically displayed on a dot matrix. The major groups correspond to well known protein families and super-families such as the protein kinases and the small GTPases. The largest cluster corresponds to proteins sharing a Dictyostelium specific FNIP repeat. Examples of ancient families such as the tRNA ligases, ABC transporters, and histidine kinases are described in more detail. The protein phosphatases and histones are two example of more recent families that are only found in eukaryotes. Recent amplifications generated families of polyketide synthases, cellulose binding proteins, and the GDT subfamily of protein kinases in Dictyostelium. Analyses of the genes and pseudogenes for the polyketide synthases and the GDTs showed the types of duplications, inversions, deletions and recombinational events that shaped Dictyostelium genome.

Chapter 5
Components of the Dictyostelium Gene Expression Regulatory Machinery
Gad Shaulsky and Eryong Huang

Control of gene expression has been recognized as a major component in the regulation of the Dictyostelium life cycle. Differential gene expression in time and in space during development and the presence of several RNA polymerases in the nucleus have indicated that Dictyostelium cells regulate gene expression like other eukaryotes. Subsequent work has shown that Dictyostelium promoters exhibit similar properties to other eukaryotic promoters and that several transcription activators bind promoters directly and regulate gene expression. Microarray data provided a broader view of the magnitude of change in developmental gene expression and solidified the notion that Dictyostelium gene expression is regulated by complex and intricate processes. In light of these observations, the analysis of the genome sequence delivered a surprising finding. Dictyostelium cells have the lowest proportion of highly conserved transcription activators of all the eukaryotes whose genomes have been sequenced, yet they regulate their genes just as well. Here, we describe the Dictyostelium homologues of the most common eukaryotic proteins that regulate gene expression, and provide a brief description of their functions. The Dictyostelium genome carries homologues of all the known RNA polymerase subunits, members of all the transcription factor gene families, and members of all but three of the transcription activator gene families (basic helix-loop-helix activators, steroid hormone receptors and fork-head transcription activators). The study of transcriptional regulation in Dictyostelium is not as advanced as it is in other eukaryotes, but the paucity of transcription activators and the unique base composition of the genome raise interesting questions that may only be answerable in Dictyostelium. The availability of the genome sequence and the parts list of the transcriptional regulatory machinery will undoubtedly lead to renewed interest and exciting discoveries in the post genomic era.

Chapter 6
Signal Transduction via G-Protein-Coupled Receptors, Trimeric G Proteins, and RGS Proteins
Dale Hereld

The completed genome sequence of Dictyostelium discoideum has revealed an impressive collection of seven-transmembrane-domain G-protein-coupled receptors (GPCR), G proteins, and Regulators of G Protein Signaling, or RGS proteins. While many of the G proteins had already been identified and, for the most part, are well characterized, it is now apparent that their functions are likely to be regulated by a comparable number of largely uncharacterized RGS proteins. Most surprising of all, the genome encodes at least 17 homologs of Family C GPCRs resembling GABA_B neurotransmitter receptors and another 25 homologs of Frizzled and Smoothened receptors, which are highly conserved mediators of metazoan development. The existence in Dictyostelium of representatives of these receptor families, previously thought to be restricted to metazoans, suggests that they arose early in eukaryotic evolution.

Chapter 7
The Microfilament System of Dictyostelium discoideum
Francisco Rivero and Ludwig Eichinger

The recent completion of the Dictyostelium discoideum genome sequence provided the basis for a genome-wide survey of the microfilament system. The analyses revealed a large number and diverse array of components of the actin cytoskeleton, i.e. actin and actin-binding proteins. Approximately half of these had not been identified previously although the actin cytoskeleton of this organism has been intensively studied for more than 25 years. The Dictyostelium genome encodes representatives of all classes of actin-binding proteins and its repertoire is most similar to metazoa followed by fungi and then plants. In this chapter we briefly introduce the major functional and structural classes of actin-binding proteins, present a catalogue of components of the microfilament system and describe their proposed cellular functions. We discuss our analyses in the context of plants, fungi and metazoa.

Chapter 8
The Small GTPase Superfamily
Gerald Weeks, Pascale Gaudet and Robert H. Insall

The small GTPases are a family of signalling molecules with roles throughout cell physiology. The Dictyostelium genome contains a surprisingly large number of small GTPases, encoded by at least 115 different genes, including members of all the important subfamilies (Ras, Rac/Rho, ARF, Rab and Ran). The actin regulators Rho and Cdc42 are not found though their absence is compensated for by a relatively large number of Racs. In general, individual family members that are conserved between metazoans are also found in Dictyostelium. This suggests that nearly all roles for GTPases in fundamental cell physiology are similar in Dictyostelium and higher eukaryotes. However, we also find a large number of additional GTPase genes without any obvious relatives in other species, which implies that a number of additional Dictyostelium-specific roles for small GTPases have emerged during evolution.

Chapter 9
The Dictyostelium Kinome: Protein Kinase Signaling Pathways that Regulate Growth and Development
Alan R. Kimmel

Protein kinase signaling regulates every phase of eukaryotic growth and development. Not surprisingly, the protein kinases comprise one of the largest and most diverse gene families in eukaryotes. In Dictyostelium, approximately 2.5% of the protein coding genes are represented by the protein kinases. This review focuses on the complexity of the Dictyostelium kinome in a comparison with other systems. In addition, specific protein kinase signaling pathways are described that regulate the major stages of development. Particular attention is devoted to the proteins kinases that organize cell polarity and chemotaxis and that regulate cell fate determination. Emphases include novel pathways involving two-component signaling and the role of tyrosine phosphorylation in an organism that exhibits unicellular growth, but multicellular development.

Chapter 10
Glycosyltransferase Genomics in Dictyostelium discoideum
Christopher M. West, Hanke van der Wel, Pedro M. Coutinho and Bernard Henrissat

The sugar chains of glycoproteins, glycolipids and polysaccharides play fundamental roles in cell physiology and development in eukaryotic microbes, as for plants and animals. Although the glycan chains of microbial glycoconjugates are very diverse, the glycosyltransferases that regulate their glycosylation have descended along evolutionary lineages that make possible the prediction of function from their sequences. From the known glycosyltransferase sequences available today and classified in the web accessible CAZy database, it is possible to search an organism's genome for potential glycosyltransferase genes and in turn make certain predictions about its glycome. Here, this approach is applied to the cellular slime mold Dictyostelium discoideum. Its 76 predicted glycosyltransferases confirm biochemical and genetic studies showing that Dictyostelium has a rich and traditional eukaryote-like glycome and, in addition, make unanticipated predictions about structural diversity. Peripheral modifications of N- and O-linked glycans in the Golgi appear to be more varied than has been surmised from global biochemical analyses, based on the prediction of ten a3/4-fucosyltransferases, thirteen predicted b2,3,4 and 6-GlcNAc-transferases, and ten glycophosphotransferase-like sequences. Predicted bifunctional diglycosyltransferases with N-terminal signal anchors suggest that up to three different secretory polysaccharides are also formed in the Golgi. Finally, new predicted soluble glycosyltransferases are candidates for multiple protein glycosylation pathways in the cytoplasm and/or nucleus.

Chapter 11
How Many Protein Encoding Genes Does Dictyostelium discoideum Have?
Rolf M. Olsen

Automatic annotation of the recently sequenced genome of Dictyostelium discoideum found 13,541 ORFs. This dataset is analyzed to estimate how many ORFs can be recognized that are unlikely to encode functional proteins (˜2600-3100) and how many can confidently be considered dispensable (˜400) to obtain a maximum number of proteins in the Dictyostelium proteome (10,000-10,500). In addition, the results of a large comparative genomics effort to identity clusters of eukaryotic orthologs are used to estimate the size of a "core" proteome which has survived over a billion years of evolution (˜2000 proteins). This "core" is supplemented by a few hundred proteins, primarily associated with aggregation and morphogenesis specific to Dictyostelium. This analysis is done within a framework for understanding duplications and deletions in genome evolution that was enunciated by Susumu Ohno more than thirty years ago and substantially validated by recent analyses of genome history following whole genome duplications.

Chapter 12
Whole-Genome Functional Analyses in Dictyostelium
Adam Kuspa

The completion of the Dictyostelium genome sequence has put the prospects for whole-genome analyses on a solid footing. The challenge now is to develop and pursue approaches that combine the power of microbial genetics and modern analytic techniques with knowledge of the genetic information to accelerate the understanding of physiological and developmental processes on a global scale. This chapter reviews current progress and future challenges of functional genomic studies in Dictyostelium.

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