African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T. brucei-specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.

TriTrypDB (http://tritrypdb.org) is an integrated database providing access to genome-scale datasets for kinetoplastid parasites, and supporting a variety of complex queries driven by research and development needs. TriTrypDB is a collaborative project, utilizing the GUS/WDK computational infrastructure developed by the Eukaryotic Pathogen Bioinformatics Resource Center (EuPathDB.org) to integrate genome annotation and analyses from GeneDB and elsewhere with a wide variety of functional genomics datasets made available by members of the global research community, often pre-publication. Currently, TriTrypDB integrates datasets from Leishmania braziliensis, L. infantum, L. major, L. tarentolae, Trypanosoma brucei and T. cruzi. Users may examine individual genes or chromosomal spans in their genomic context, including syntenic alignments with other kinetoplastid organisms. Data within TriTrypDB can be interrogated utilizing a sophisticated search strategy system that enables a user to construct complex queries combining multiple data types. All search strategies are stored, allowing future access and integrated searches. ‘User Comments’ may be added to any gene page, enhancing available annotation; such comments become immediately searchable via the text search, and are forwarded to curators for incorporation into the reference annotation when appropriate.

In trypanosomes there is an almost total reliance on posttranscriptional mechanisms to alter gene expression; here, heat shock was used to investigate the response to an environmental signal. Heat shock rapidly and reversibly induced a decrease in polysome abundance, and the consequent changes in mRNA metabolism were studied. Both heat shock and polysome dissociation were necessary for (1) a reduction in mRNA levels that was more rapid than normal turnover, (2) an increased number of P-body-like granules that contained DHH1, SCD6 and XRNA, (3) the formation of stress granules that remained largely separate from the P-body-like granules and localise to the periphery of the cell and, (4) an increase in the size of a novel focus located at the posterior pole of the cell that contain XRNA, but neither DHH1 nor SCD6. The response differed from mammalian cells in that neither the decrease in polysomes nor stress-granule formation required phosphorylation of eIF2α at the position homologous to that of serine 51 in mammalian eIF2α and in the occurrence of a novel XRNA-focus.Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/18/3002/DC1 Key words: Heat shock, Trypanosoma brucei, Stress granules, eIF2 alpha, P-bodies SummaryHeat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2α phosphorylation at Thr169 Journal of Cell Science 3003 Heat shock stress granules and P-bodies in trypanosomes from a single promoter (Johnson et al., 1987; Kooter et al., 1987;Martinez-Calvillo et al., 2004;Martinez-Calvillo et al., 2003), and monocistronic mRNAs result from trans-splicing of a short, capped leader to the 5Ј end and linked 3Ј cleavage and polyadenylation of the upstream mRNA (Campbell et al., 1984;LeBowitz et al., 1993;Liang et al., 2003;Matthews et al., 1994;Schürch et al., 1994;Ullu et al., 1993). Consequently, the regulation of gene expression in trypanosomes is predominantly post-transcriptional (Clayton and Shapira, 2007). There is evidence for the presence of P-bodies: DHH1, XRNA and one Pumilio-family protein are located in cytoplasmic granules in normally growing cells (Caro et al., 2006;Cassola et al., 2007;Dallagiovanna et al., 2007;Dallagiovanna et al., 2005;Holetz et al., 2007), although any role in the regulation of gene expression has yet to be determined. In contrast to mRNA synthesis, the mechanisms of translation initiation and elongation appear to be typical for a eukaryote; all the factors identified in metazoa and yeast are present in the trypanosome genome (Ivens et al., 2005), although a functional analysis has only been performed on a small number (Dhalia et al., 2006;Dhalia et al., 2005) and very little is known about regulation of translation (Clayton and Shapira, 2007).Little is known about how the overall rate of gene expression is regulated, for example when trypanosomes stop growth and enter stationary phase. The only such phenomenon that has been investigated in any detail is the response to heat sh...

Significance African trypanosomes are parasites that can cause African sleeping sickness in humans. Humans and some primates, but not other mammals, have a gene called APOL1 that protects against certain trypanosomes. Genetic variants in APOL1 that arose in Africa are strongly associated with kidney disease in African Americans. These kidney disease-associated variants may have risen to high frequency in Africa because they can defend humans against a particularly pathogenic trypanosome. In this paper, we show how APOL1 has evolved by analyzing the distribution of these variants in Africa and then elucidating the molecular mechanisms that enhance their trypanosome killing capacity. We also show that these antitrypanosomal APOL1 variants may have adverse consequences for the host.

Trypanosomes are important disease agents and excellent models for the study of evolutionary cell biology. The trypanosome flagellar pocket is a small invagination of the plasma membrane where the flagellum exits the cytoplasm and participates in many cellular processes. It is the only site of exocytosis and endocytosis and part of a multiorganelle complex that is involved in cell polarity and cell division. Several flagellar pocket-associated proteins have been identified and found to contribute to trafficking and virulence. In this Review we discuss the contribution of the flagellar pocket to protein trafficking, immune evasion and other processes.

Two B-type cyclins, B1 and B2, have been identified in mammals. Proliferating cells express both cyclins, which bind to and activate p34 cdc2 . To test whether the two B-type cyclins have distinct roles, we generated lines of transgenic mice, one lacking cyclin B1 and the other lacking cyclin B2. Cyclin B1 proved to be an essential gene; no homozygous B1-null pups were born. In contrast, nullizygous B2 mice developed normally and did not display any obvious abnormalities. Both male and female cyclin B2-null mice were fertile, which was unexpected in view of the high levels and distinct patterns of expression of cyclin B2 during spermatogenesis. We show that the expression of cyclin B1 overlaps the expression of cyclin B2 in the mature testis, but not vice versa. Cyclin B1 can be found both on intracellular membranes and free in the cytoplasm, in contrast to cyclin B2, which is membrane-associated. These observations suggest that cyclin B1 may compensate for the loss of cyclin B2 in the mutant mice, and implies that cyclin B1 is capable of targeting the p34 cdc2 kinase to the essential substrates of cyclin B2.Mitotic B-type cyclins activate the p34 cdc2 protein kinase to form maturation promoting factor, which is required for cells to undergo mitosis (1-3). Only two B-type cyclins, B1 (4, 5) and B2 (6, 7), have been identified so far in mammals, although chickens, frogs, flies, and nematode worms possess a third, more distant relative-cyclin B3 (8, 9). The genes of cyclin B1 and B2 show very little similarity in the first 100 residues, and about 57% identity in the remaining 300 residues. The two genes must have diverged early in vertebrate evolution, because frogs have both genes. The family resemblances are strongly conserved: Goldfish B1 is almost 70% identical to mouse B1 in the C-terminal 300 residues, and chicken B2 is 75% identical to mouse B2 in the equivalent region. Fig. 1 shows a dendrogram of the current B-type cyclin family tree, omitting yeast examples that cluster together as a separate branch. Cyclins B1 and B2 are coexpressed in the majority of dividing cells, although their subcellular localization differs, with cyclin B1 usually associated with microtubules, and cyclin B2 with intracellular membranes (10-15). Cyclin B1 enters the nucleus in late G 2 phase of the cell cycle, whereas cyclin B2 does not (15). Cyclins B1 and B2 also show different patterns of expression during murine spermatogenesis (6), and in Xenopus oocytes, cyclin B2 protein is already present in unactivated oocytes, whereas cyclin B1 is not synthesized until progesterone induced oocyte maturation (16,17). These considerations would lead one to suspect that cyclins B1 and B2 have specialized roles in preparing cells for mitosis, but there is no direct evidence for this view from studies in vertebrates.

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