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...

Erythromycin A, a clinically important polyketide antibiotic, is produced by the Gram-positive bacterium Saccharopolyspora erythraea. In an arrangement that seems to be generally true of antibiotic biosynthetic genes in Streptomyces and related bacteria like S. erythraea, the ery genes encoding the biosynthetic pathway to erythromycin are clustered around the gene (ermE) that confers self-resistance on S. erythraea. The aglycone core of erythromycin A is derived from one propionyl-CoA and six methylmalonyl-CoA units, which are incorporated head-to-tail into the growing polyketide chain, in a process similar to that of fatty-acid biosynthesis, to generate a macrolide intermediate, 6-deoxyerythronolide B. 6-Deoxyerythronolide B is converted into erythromycin A through the action of specific hydroxylases, glycosyltransferases and a methyltransferase. We report here the analysis of about 10 kilobases of DNA from S. erythraea, cloned by chromosome 'walking' outwards from the erythromycin-resistance determinant ermE, and previously shown to be essential for erythromycin biosynthesis. Partial sequencing of this region indicates that it encodes the synthase. Our results confirm this, and reveal a novel organization of the erythromycin-producing polyketide synthase, which provides further insight into the mechanism of chain assembly.

Background-Endothelium-derived nitric oxide (NO) is synthesised from L-arginine by endothelial nitric oxide synthase (eNOS) encoded by the NOS 3 gene on chromosome 7. Because reduced NO synthesis has been implicated in the development of coronary atherosclerosis, which has a heritable component, we hypothesised that polymorphisms of NOS 3 might be associated with increased susceptibility to this disorder. Methods and Results-Single-strand conformation polymorphism analysis of NOS 3 identified a G3 T polymorphism in exon 7 of the gene which encodes a Glu3 Asp amino acid substitution at residue 298 of eNOS. We investigated the relationship between this Glu

The macrocyclic polyketides rapamycin and FK506 are potent immunosuppressants that prevent T-cell proliferation through specific binding to intracellular protein receptors (immunophilins). The cloning and specific alteration of the biosynthetic genes for these polyketides might allow the biosynthesis of clinically valuable analogues. We report here that three clustered polyketide synthase genes responsible for rapamycin biosynthesis in Streptomyces hygroscopicus together encode 14 homologous sets of enzyme activities (modules), each catalyzing a specific round of chain elongation. An adjacent gene encodes a pipecolate-incorporating enzyme, which completes the macrocycle. The total of 70 constituent active sites makes this the most complex multienzyme system identiried so far. The DNA region sequenced (107.3 kbp) contains 24 additional open reading frames, some of which code for proteins governing other key steps in rapamycin biosynthesis.Polyketides are a large and highly diverse group of natural products that includes antibiotics, antitumor compounds, and immunosuppressants. The specific binding of polyketides to prevent T-cell proliferation was reported in 1992 by Schreiber (1) and Rosen and Schreiber (2). These polyketide metabolites are produced by successive condensation of simple carboxylic acid units (primarily acetate and propionate) as for fatty acid biosynthesis (3), except that the 3-keto function introduced during each elongation cycle may be reduced only partially or not at all. Macrocyclic polyketides are produced principally by Streptomyces and related filamentous bacteria, through the action of so-called type I modular polyketide synthases (PKSs), multienzymes in which different sets (modules) of enzymic activities catalyze each successive round of elongation, as first shown for the erythromycin-producing PKS (4-6). Characterization and genetic engineering of such systems to produce "hybrid" products (7) are particularly challenging because of the large size of the genes and their products and because the factors that control the specificity of chain extension are still largely unknown (7,8).Rapamycin ( Fig. 1) is a macrocyclic polyketide from Streptomyces hygroscopicus that, in addition to its antifungal (13) and antitumor (14) properties, is a potent immunosuppressant (15). Like the structurally related FK506, rapamycin is of interest for the clinical treatment of autoimmune disease (16) and in the prevention of rejection of organ and skin allografts (15,17). In spite of their similar polyketide backbone, these immunosuppressants act in radically different ways on T cells, FK506 by inhibiting the production of interleukin 2 (1, 2) and rapamycin by preventing the proliferative response to interleukin 2 bound at the interleukin 2 receptor (18). The engineered biosynthesis of altered rapamycins would also be of great interest for the study of these signaling processes. We have therefore undertaken a detailed study of the organization of the rapamycin biosynthetic genes in S. hygroscopicus....