Basal body multipotency and axonemal remodelling are two pathways to a 9+0 flagellum.

Eukaryotic cilia/flagella exhibit two characteristic ultrastructures reflecting two main functions; a 9+2 axoneme for motility and a 9+0 axoneme for sensation and signalling. Whether, and if so how, they interconvert is unclear. Here we analyse flagellum length, structure and molecular composition changes in the unicellular eukaryotic parasite Leishmania during the transformation of a life cycle stage with a 9+2 axoneme (the promastigote) to one with a 9+0 axoneme (the amastigote). We show 9+0 axonemes can be generated by two pathways: by de novo formation and by restructuring of existing 9+2 axonemes associated with decreased intraflagellar transport. Furthermore, pro-basal bodies formed under conditions conducive for 9+2 axoneme formation can form a 9+0 axoneme de novo. We conclude that pro-centrioles/pro-basal bodies are multipotent and not committed to form either a 9+2 or 9+0 axoneme. In an alternative pathway structures can also be removed from existing 9+2 axonemes to convert them to 9+0.

System dynamic modelling to assess economic viability and risk trade-offs for ecological restoration in South Africa.

Can markets assist by providing support for ecological restoration, and if so, under what conditions? The first step in addressing this question is to develop a consistent methodology for economic evaluation of ecological restoration projects. A risk analysis process was followed in which a system dynamics model was constructed for eight diverse case study sites where ecological restoration is currently being pursued. Restoration costs vary across each of these sites, as do the benefits associated with restored ecosystem functioning. The system dynamics model simulates the ecological, hydrological and economic benefits of ecological restoration and informs a portfolio mapping exercise where payoffs are matched against the likelihood of success of a project, as well as a number of other factors (such as project costs and risk measures). This is the first known application that couples ecological restoration with system dynamics and portfolio mapping. The results suggest an approach that is able to move beyond traditional indicators of project success, since the effect of discounting is virtually eliminated. We conclude that systems dynamic modelling with portfolio mapping can guide decisions on when markets for restoration activities may be feasible.

Genome organization is a major component of gene expression control in response to stress and during the cell division cycle in trypanosomes.

The trypanosome genome is characterized by RNA polymerase II-driven polycistronic transcription of protein-coding genes. Ten to hundreds of genes are co-transcribed from a single promoter; thus, selective regulation of individual genes via initiation is impossible. However, selective responses to external stimuli occur and post-transcriptional mechanisms are thought to account for all temporal gene expression patterns. We show that genes encoding mRNAs that are differentially regulated during the heat-shock response are selectively positioned in polycistronic transcription units; downregulated genes are close to transcription initiation sites and upregulated genes are distant. We demonstrate that the position of a reporter gene within a transcription unit is sufficient to reproduce this effect. Analysis of gene ontology annotations reveals that positional bias is not restricted to stress-response genes and that there is a genome-wide organization based on proximity to transcription initiation sites. Furthermore, we show that the relative abundance of mRNAs at different time points in the cell division cycle is dependent on the location of the corresponding genes to transcription initiation sites. This work provides evidence that the genome in trypanosomes is organized to facilitate co-coordinated temporal control of gene expression in the absence of selective promoters.

A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor.

Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei. This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei, including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes.

Archaeal phylogenomics provides evidence in support of a methanogenic origin of the Archaea and a thaumarchaeal origin for the eukaryotes.

We have developed a machine-learning approach to identify 3537 discrete orthologue protein sequence groups distributed across all available archaeal genomes. We show that treating these orthologue groups as binary detection/non-detection data is sufficient to capture the majority of archaeal phylogeny. We subsequently use the sequence data from these groups to infer a method and substitution-model-independent phylogeny. By holding this phylogeny constrained and interrogating the intersection of this large dataset with both the Eukarya and the Bacteria using Bayesian and maximum-likelihood approaches, we propose and provide evidence for a methanogenic origin of the Archaea. By the same criteria, we also provide evidence in support of an origin for Eukarya either within or as sisters to the Thaumarchaea.

Characterization and differential nuclear localization of Nopp140 and a novel Nopp140-like protein in trypanosomes.

Trypanosomatids possess two homologues of Nopp140: a canonical Nopp140 and a Nopp140-like protein (TbNoLP) in which a GAR domain replaces the C-terminal SRP40 domain. Both are phosphorylated and coimmunoprecipitate with RNA polymerase I. Each paralogue has a distinct subnuclear localization, and depletion of TbNoLP produces an enlarged nucleolus in which TbNopp140-containing regions disperse. The restricted occurrence pattern of NoLP proteins reflects an intriguing convergence in evolution, suggestive of a function in nucleoplasmic small nucleolar ribonucleoprotein shuttling.

An in silico analysis of trypanosomatid RNA polymerases: insights into their unusual transcription.

African trypanosomes employ both Pol I (RNA polymerase I) and Pol II to transcribe protein-coding genes in large polycistronic units of up to 50 genes. Subsequent processing produces mature capped mRNAs. Evidence suggests that regulation of gene expression is primarily exerted post-transcriptionally. Here, we use the recently completed genome sequences of three trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, in an in silico analysis of their fundamental RNA polymerase complexes. The core complement of Pol II subunits, including those that are shared with Pol I and Pol III are present. However, both Pol I and Pol III complexes are missing members of the rpoE-rpoF subunit groups. Out of the five shared subunits, both RPB5 and RPB6 have two isoforms in the three trypanosomes. One represents the canonical polymerase subunit and the other differs by insertion or deletion of stretches of charged residues. We propose that these alternative isoforms function in distinct polymerase complexes, and may influence recruitment of the trypanosome RPB4-RPB7 heterodimer.

The identification of circular extrachromosomal DNA in the nuclear genome of Trypanosoma brucei.

Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei.

The cell biology of parasitism in Trypanosoma brucei: insights and drug targets from genomic approaches?

The African trypanosome, Trypanosoma brucei exhibits a complex, digenetic life cycle that alternates between the tsetse fly vector and the mammalian host. The life cycle is characterised by a complex series of cell type differentiations and variations in metabolism. In addition the trypanosome exhibits a particular cell biology that has become adapted for its role as a parasite. This article places some of these areas in a frame-work that considers the role of cellular processes in parasitism. I rehearse some conclusions from recent studies and provide hyphotheses and suggestions for future work. Areas debated include: cell surface protein expression, cell differentiation, endomembrane trafficking and protein targeting, the cytoskeleton,flagellum functions in motility, attachment and plasma membrane differentiation, organelle specialisations, control of cell cycle, parasite/host, parasite/parasite and parasite/vector interactions. The review also focuses on the likely impact of the genome project and reverse genetics in providing greater insight to these cellular processes and how, if coordinated with some alan by scientists and funding agencies, this may provide novel targets for future drug development.

A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei.

In the mammalian host, African trypanosomes generate consecutive waves of parasitaemia by changing their antigenic coat. Because this coat consists of a single type of variant surface glycoprotein (VSG), the question arises of how a trypanosome accomplishes the transcription of only one of a multi-allelic family of VSG expression site loci to display a single VSG type on the surface at any one time. No major differences have been detected between the single active expression site and the cohort of inactive expression sites. Here we identify an extranucleolar body containing RNA polymerase I (pol I) that is transcriptionally active and present only in the bloodstream form of the parasite. Visualization of the active expression site locus by tagging with green fluorescent protein shows that it is specifically located at this unique pol I transcriptional factory. The presence of this transcriptional body in postmitotic nuclei and its stability in the nucleus after DNA digestion provide evidence for a coherent structure. We propose that the recruitment of a single expression site and the concomitant exclusion of inactive loci from a discrete transcriptional body define the mechanism responsible for VSG mono-allelic expression.

The extended tubulin superfamily.

Although most eukaryotic cells can express multiple isotypes of alphabeta-tubulin, the significance of this diversity has not always been apparent. Recent data indicate that particular alphabeta-tubulin isotypes, both genome encoded and those derived by post-translational modification, can directly influence microtubule structure and function--thus validating ideas originally proposed in the multitubulin hypothesis over 25 years ago. It has also become increasingly evident over the past year that some (but intriguingly not all) eukaryotes encode several other tubulin proteins, and to date five further members of the tubulin superfamily, gamma, delta, epsilon, zeta and eta, have been identified. Although the role of gamma-tubulin in the nucleation of microtubule assembly is now well established, far less is known about the functions of delta-, epsilon-, zeta- and eta-tubulin. Recent work has expanded our knowledge of the functions and localisation of these newer members of the tubulin superfamily, and the emerging data suggesting a restricted evolutionary distribution of these 'new' tubulin proteins, conforms to established knowledge of microtubule cell biology. On the basis of current evidence, we predict that delta-, epsilon-, zeta- and eta-tubulin all have functions associated with the centriole or basal body of eukaryotic cells and organisms.

A trypanosome structure involved in transmitting cytoplasmic information during cell division.

African trypanosomes are protozoan parasites that cause sleeping sickness in humans through a tsetse fly vector. The procyclic form of Trypanosoma brucei has a single, attached flagellum that describes a helical path along the cell from posterior to anterior. During division, a specific flagellum-flagellum connection is elaborated between the new and old flagellum. This connector was present only during cell duplication and was found to be involved in the replication of the helical cell pattern and polarity. This finding implicates the concept of cytotaxis in cell morphogenesis in trypanosomes.

Limitation of Trypanosoma brucei parasitaemia results from density-dependent parasite differentiation and parasite killing by the host immune response.

In the bloodstream of its mammalian host, the "slender" form of Trypanosoma brucei replicates extracellularly, producing a parasitaemia. At high density, the level of parasitaemia is limited at a sublethal level by differentiation to the non-replicative "stumpy" form and by the host immune response. Here, we derive continuous time equations to model the time-course, cell types and level of trypanosome parasitaemia, and compare the best fits with experimental data. The best fits that were obtained favour a model in which both density-dependent trypanosome differentiation and host immune response have a role in limiting the increase of parasites, much poorer fits being obtained when differentiation and immune response are considered independently of one another. Best fits also favour a model in which the slender-to-stumpy differentiation progresses in a manner that is essentially independent of the cell cycle. Finally, these models also make the prediction that the density-dependent trypanosome differentiation mechanism can give rise to oscillations in parasitaemia level. These oscillations are independent of the immune system and are not due to antigenic variation.

Protist tubulins: new arrivals, evolutionary relationships and insights to cytoskeletal function.

The protists exhibit probably the most extravagant expression of microtubule-containing structures found in any organism. These structures--flagella, cilia, axostyles, spindles and a veritable constellation of microtubule bundles and cortical arrays--provide shape, form, motility, anchorage and apparatuses for feeding. The cytoskeletal structures have a precise order (i.e. size, position and number) that must be replicated and segregated with fidelity at each division, some components being inherited conservatively and others semi-conservatively. Intriguingly, it is now apparent that much of the high-order organisation, which was recognised and described by light and electron microscopy during the last century, is a reflection of molecular polarities set by assembly of constituent proteins. Tubulins and microtubules lie at the heart of this morphogenetic pattern.

CAP5.5, a life-cycle-regulated, cytoskeleton-associated protein is a member of a novel family of calpain-related proteins in Trypanosoma brucei.

The cell shape of African trypanosomes is determined by the presence of an extensive subpellicular microtubule cytoskeleton. Other possible functions of the cytoskeleton, such as providing a potential framework for signalling proteins transducing information from the intracellular and extracellular environment, have not yet been investigated in trypanosomes. In this study, we have identified a novel cytoskeleton-associated protein in Trypanosoma brucei. CAP5.5 is the first member of a new family of proteins in trypanosomes, characterised by their similarity to the catalytic region of calpain-type proteases. CAP5.5 is only expressed in procyclic, but not in bloodstream, trypanosomes. Furthermore, CAP5.5 has been shown to be both myristoylated and palmitoylated, suggesting a stable interaction with the cell membrane. A bioinformatics analysis of the trypanosome genome revealed a diverse family of calpain-related proteins with primary structures similar to CAP5.5, but of varying length. We suggest a nomenclature for this new family of proteins in T. brucei.

The biology of kinetoplastid parasites: insights and challenges from genomics and post-genomics.

Kinetoplastid parasites exhibit a rich and diverse biology which mirrors many of the most interesting topics of current interest and study in the broader biological sciences. These evolutionarily ancient organisms possess intriguing mechanisms for control of gene expression, and exhibit complex patterns of cell morphogenesis orchestrated by an internal cytoskeleton. Their cell shapes change during a set of complex cell type differentiations in their life cycles. These differentiations are intimately linked to interactions with mammalian hosts or insect vectors, and often, these differentiations appear central to the successful transfer of the parasite between vector and host, and host and vector. The basics of this rich and complex cell and life cycle biology were described (with often rather forgotten clarity and prescience) in the early period of the last century. The last 30 years have seen major developments in our understanding of this biology. Ultrastructural differences in the various cells of the life cycle stages of Trypanosoma brucei, Trypanosoma cruzi and the various Leishmania species have been documented, and such studies have proven highly informative in defining important aspects of parasite adaptation. They have also proven to be a rich source of information for defining unusual aspects of parasite cell biology, novel organelles and cell architecture. This ultrastructural cell biology has been mirrored in a set of biochemical explanations defining unusual aspects of metabolism, surface molecules, and organelles. Finally, the application of molecular biology to these parasites revealed fascinating layers of complexity in the control of gene expression. These molecular studies have given us particular insights into polycistronic transcription, trans-splicing, RNA editing and gene rearrangements during antigenic variation. In contrast to other microbial systems, these cell biological, biochemical and molecular studies have not been greatly aided by insights gained from genetics--the diploid nature of the genome has discouraged the application of selectional genetics, mutant isolation and analysis. This is an important fact, since in general, it means that we have only recently started to analyse the phenotypes of mutants produced in the context of reverse genetics. In the following, I will argue that this lack of investment in the analysis of mutant phenotype is just one of the challenges that will need to be met if we are to gain the expected added value from the parasite genome projects. In this presentation, I will use some of the current areas of interest in the biology of T. brucei, T. cruzi and the Leishmania species to rehearse some of the insights and challenges that are likely to stem from the application of genomics and post-genomic studies to the kinetoplastid parasites. In some cases, I will exemplify points by illustrations from my laboratory's work, interests and hypotheses. The presentation slants therefore towards T. brucei biology, however, in each case the reader will, no doubt, see the generalities of application to other kinetoplastid parasites.

Diversity and dynamics of the minichromosomal karyotype in Trypanosoma brucei.

The genome of African trypanosomes contains a large number of minichromosomes. Their only proposed role is in the expansion of the parasites' repertoire of telomeric variant surface glycoprotein (VSG) genes as minichromosomes carry silent VSG gene copies in telomeric locations. Despite their importance as VSG gene donors, little is known about the actual composition of the minichromosomal karyotype and the stability of its inheritance. In this study we show, by using high-resolution pulsed-field electrophoresis, that a non-clonal trypanosome population contains an extremely diverse pattern of minichromosomes, which can be resolved into less complex clone-specific karyotypes by non-selective cloning. We show that the minichromosome patterns of such clones are stable over at least 360 generations. Furthermore, using DNA markers for specific minichromosomes, we demonstrate the mitotic stability of these minichromosomes within the population over a period of more than 5 years. Length variation is observed for an individual minichromosome and is most likely caused by a continuous telomeric growth of approximately 6 bp per telomere per cell division. This steady telomeric growth, counteracted by stochastic large losses of telomeric sequences is the most likely cause of minichromosome karyotype heterogeneity within a population.

Anisomorphic cell division by African trypanosomes.

In the bloodstream of a mammalian host, African trypanosomes are pleomorphic; the shorter, non-proliferative, stumpy forms arise from longer, proliferative, slender forms with differentiation occurring via a range of morphological intermediates. In order to investigate how the onset of morphological change is co-ordinated with exit from the cell cycle we first characterized slender form cell division. Outgrowth of the new flagellum was found to occur at a linear rate, so by using outgrowth of the new flagellum as a temporal marker of the cell cycle we were able determine the order in which single copy organelles (nucleus, kinetoplast and mitochondrion) were segregated. We also found that flagellar length was an effective marker of the slender to stumpy differentiation and were, therefore, able to study both cell division and differentiation. When these differentiating cells were compared to cells undergoing proliferative cell division, they were found to be anisomorphic--showing discernible differences not only in the length of their new flagella but also in the shape and size of the cells and their nuclei.

Targeting of cytoskeletal proteins to the flagellum of Trypanosoma brucei.

The eukaryotic flagellum represents one of the most complex macromolecular structures found in any organism and contains more than 250 proteins. Due to the relative ease of genetic manipulation the flagellum of Trypanosoma brucei has emerged as an accessible model system to study the morphogenesis and dynamics of this organelle. We have recently started to characterise the mechanisms by which components of the cytoskeletal fraction of the flagellum, such as the axoneme, the paraflagellar rod and the flagellar attachment zone, are targeted by proteins synthesised in the cytoplasm and assembled. Here, we present the identification of a novel actin-related protein as a component of the axoneme. We show that this protein shares the tripeptid motif histidine-leucine-alanine (HLA) with one of the major proteins of the paraflagellar rod, PFRA. Building on previous work from this lab which showed that a deletion comprising this motif abolished targeting of PFRA to the flagellum we demonstrate in this study that the deletion of the tripeptid motif is sufficient to achieve mistargeting both of the PFRA and the actin-related protein. We propose that this motif represents an essential part of a flagellar targeting machinery in trypanosomes and possibly in other flagellated organisms.

Genomics and post-genomics in parasitology: genome babble or a real opportunity?

The genome projects represent one of the most important developments in our knowledge of parasites. However, translation of this knowledge into an understanding of parasite biology and then on to drugs, vaccines and other healthcare developments for the diseases will need some élan and clarity of thought by scientists and funding organizations. Only then will the activity associated with post-genomics be turned from what I have termed 'genome babble' to real opportunities in understanding these parasites.