Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself.

Background: In membrane trafficking, the mechanisms ensuring vesicle fusion specificity remain to be fully elucidated. Early models proposed that specificity was encoded entirely by SNARE proteins; more recent models include contributions from Rab proteins, Syntaxin-binding (SM) proteins and tethering factors. Most information on membrane trafficking derives from an evolutionarily narrow sampling of model organisms. However, considering factors from a wider diversity of eukaryotes can provide both functional information on core systems and insight into the evolutionary history of the trafficking machinery. For example, the major Qa/syntaxin SNARE families are present in most eukaryotic genomes and likely each evolved via gene duplication from a single ancestral syntaxin before the existing eukaryotic groups diversified. This pattern is also likely for Rabs and various other components of the membrane trafficking machinery.

SummaryIntracellular trafficking and protein sorting are mediated by various protein complexes, with the retromer complex being primarily involved in retrograde traffic from the endosome or lysosome to the Golgi complex. Here, comparative genomics, cell biology and phylogenetics were used to probe the early evolution of retromer and its function. Retromer subunits Vps26, Vps29 and Vps35 are near universal, and, by inference, the complex was an ancient feature of eukaryotic cells. Surprisingly, we found DSCR3, a Vps26 paralogue in humans associated with Down's syndrome, in at least four eukaryotic supergroups, implying a more ancient origin than previously suspected. By contrast, retromer cargo proteins showed considerable interlineage variability, with lineage-specific and broadly conserved examples found. Vps10 trafficking probably represents an ancestral role for the complex. Vps5, the BAR-domaincontaining membrane-deformation subunit, was found in diverse eukaryotes, including in the divergent eukaryote Trypanosoma brucei, where it is the first example of a BAR-domain protein. To determine functional conservation, an initial characterisation of retromer was performed in T. brucei; the endosomal localisation and its role in endosomal targeting are conserved. Therefore retromer is identified as a further feature of the sophisticated intracellular trafficking machinery of the last eukaryotic common ancestor, with BAR domains representing a possible third independent mechanism of membrane-deformation arising in early eukaryotes.

BackgroundTrypanosomatids utilise polycistronic transcription for production of the vast majority of protein-coding mRNAs, which operates in the absence of gene-specific promoters. Resolution of nascent transcripts by polyadenylation and trans-splicing, together with specific rates of mRNA turnover, serve to generate steady state transcript levels that can differ in abundance across several orders of magnitude and can be developmentally regulated. We used a targeted oligonucleotide microarray, representing the strongly developmentally-regulated T. brucei membrane trafficking system and ~10% of the Trypanosoma brucei genome, to investigate both between-stage, or differentiation-dependent, transcriptome changes and within-stage flexibility in response to various challenges.Results6% of the gene cohort are developmentally regulated, including several small GTPases, SNAREs, vesicle coat factors and protein kinases both consistent with and extending previous data. Therefore substantial differentiation-dependent remodeling of the trypanosome transcriptome is associated with membrane transport. Both the microarray and qRT-PCR were then used to analyse transcriptome changes resulting from specific gene over-expression, knockdown, altered culture conditions and chemical stress. Firstly, manipulation of Rab5 expression results in co-ordinate changes to clathrin protein expression levels and endocytotic activity, but no detectable changes to steady-state mRNA levels, which indicates that the effect is mediated post-transcriptionally. Secondly, knockdown of clathrin or the variant surface glycoprotein failed to perturb transcription. Thirdly, exposure to dithiothreitol or tunicamycin revealed no evidence for a classical unfolded protein response, mediated in higher eukaryotes by transcriptional changes. Finally, altered serum levels invoked little transcriptome alteration beyond changes to expression of ESAG6/7, the transferrin receptor.ConclusionWhile trypanosomes regulate mRNA abundance to effect the major changes accompanying differentiation, a given differentiated state appears transcriptionally inflexible. The implications of the absence of a transcriptome response in trypanosomes for both virulence and models of life cycle progression are discussed.

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.

Turbulence has been shown to alter different aspects of the physiology of some dinoflagellates. The response appears to be species-specific and dependent on the experimental design and setup used to generate small-scale turbulence. We examined the variability of the response of three dinoflagellate species to the turbulence, following the same experimental design used by Berdalet (1992) on Akashiwo sanguinea (Hirasaka) Ge. Hansen et Moestrup (=Gymnodinium nelsonii G. W. Martin). In all experiments, turbulence was generated by an orbital shaker at 100 rpm, which corresponded on bulk average, to dissipation rates (e, quantified using an acoustic Doppler velocimeter) of %2 cm 2 AE s )3 . Turbulence did not appreciably affect Gymnodinium sp., a small dinoflagellate. However, Alexandrium minutum Halim and Prorocentrum triestinum J. Schiller exhibited a reduced net growth rate (33% and 28%, respectively) when shaken during the exponential growth phase. Compared to the still cultures, the shaken treatments of A. minutum and P. triestinum increased the mean cell volume (up to 1.4-and 2.5-fold, respectively) and the mean DNA content (up to 1.8-and 5.3-fold, respectively). Cultures affected by turbulence recovered their normal cell properties when returned to still conditions. The swimming speed of the cells exposed to agitation was half that of the unshaken ones. Overall, the response of A. minutum and P. triestinum was similar, but with lower intensity, to that observed previously on A. sanguinea. We found no clear trends related to taxonomy or morphology.

We discuss the suggestion that differences in the nucleotide composition between plastid and nuclear genomes may provide a selective advantage in the transposition of genes from plastid to nucleus. We show that in the adenine, thymine (AT)-rich genome of Borrelia burgdorferi several genes have an AT-content lower than the average for the genome as a whole. However, genes whose plant homologues have moved from plastid to nucleus are no less AT-rich than genes whose plant homologues have remained in the plastid, indicating that both classes of gene are able to support a high AT-content. We describe the anomalous organization of dinoflagellate plastid genes. These are located on small circles of 2-3 kbp, in contrast to the usual plastid genome organization of a single large circle of 100-200 kbp. Most circles contain a single gene. Some circles contain two genes and some contain none. Dinoflagellate plastids have retained far fewer genes than other plastids. We discuss a similarity between the dinoflagellate minicircles and the bacterial integron system.

Trypanosomes are members of the kinetoplastida, a group of divergent protozoan parasites responsible for considerable morbidity and mortality worldwide. These organisms have highly complex life cycles requiring modification of their cell surface together with engagement of immune evasion systems to effect survival; both processes intimately involve the membrane trafficking system. The completion of three trypanosomatid and several additional protist genomes in the last few years is providing an exciting opportunity to evaluate, at the molecular level, the evolution and diversity of membrane trafficking across deep evolutionary time as well as to analyse in unprecedented detail the membrane trafficking systems of trypanosomes.