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Aurora-B kinase is a chromosomal passenger protein essential for chromosome segregation and cytokinesis. In the procyclic form of Trypanosoma brucei, depletion of an aurora-B kinase homologue TbAUK1 inhibited spindle formation, mitosis, cytokinesis, and organelle replication without altering cell morphology. In the present study, an RNA interference knockdown of TbAUK1 or overexpression of inactive mutant TbAUK1-K58R in the bloodstream form also resulted in defects in spindle formation, chromosome segregation, and cytokinesis but allowed multiple rounds of nuclear DNA synthesis, nucleolus multiplication, and continuous replication of kinetoplast, basal body, and flagellum. The typical trypanosome morphology was lost to an enlarged round shape filled with microtubules. It is thus apparent that there are distinctive mechanisms of action of TbAUK1 in regulating cell division between the two developmental stages of trypanosome. While it exerts a tight control on mitosis, organelle replication, and cytokinesis in the procyclic form, it regulates cytokinesis without rigid control over either nuclear DNA synthesis or organelle replication in the bloodstream form. The molecular basis underlining these discrepancies remains to be explored.Trypanosoma brucei is an ancient unicellular eukaryote whose propagation involves a cyclic transmission between the mammalian host and the insect vector tsetse fly. A trypanosome cell contains a single flagellum, basal body, nucleus, mitochondrion, and the mitochondrial DNA complex known as the kinetoplast. Each of these organelles/structures is replicated and segregated into the two daughter cells in a wellsynchronized manner during cell division, implying a delicate coordination among these events (25). Other than the usual sequential G 1 , S, G 2 , and M phases, there is also an S phase for the kinetoplast completed prior to the nuclear S phase (45). The kinetoplast is replicated prior to mitosis and closely associated with the flagellum basal body, with its segregation dependent on the separation of replicated basal bodies, leading the cell through cytokinesis and eventual cell division in good coordination with nuclear division (30).Treatment of the procyclic form of T. brucei cells with okadaic acid prevented kinetoplast/basal body segregation and resulted in multinucleated cells, suggesting a normal progression of mitosis in cells defective in cytokinesis, which is controlled largely by the kinetoplast cycle (7). Treatment with aphidicolin or rhizoxin (29) or RNA interference (RNAi) silencing of the G 1 or mitotic cyclins (15, 24) or the cdc2-related kinases (CRKs) (37) in the procyclic-form cells resulted in inhibition of nuclear division but not kinetoplast/basal body segregation and subsequent cytokinesis, resulting in anucleate daughter cells designated the zoids. These results suggest that the checkpoint linking mitosis with cytokinesis may be weak or absent in the procyclic form of T. brucei and that kinetoplast/ basal body segregation alone is sufficient to drive some of t...

Aurora B kinase is an essential regulator of chromosome segregation with the action well characterized in eukaryotes. It is also implicated in cytokinesis, but the detailed mechanism remains less clear, partly due to the difficulty in separating the latter from the former function in a growing cell. A chemical genetic approach with an inhibitor of the enzyme added to a synchronized cell population at different stages of the cell cycle would probably solve this problem. In the deeply branched parasitic protozoan Trypanosoma brucei, an Aurora B homolog, TbAUK1, was found to control both chromosome segregation and cytokinetic initiation by evidence from RNAi and dominant negative mutation. To clearly separate these two functions, VX-680, an inhibitor of TbAUK1, was added to a synchronized T. brucei procyclic cell population at different cell cycle stages. The unique trans-localization pattern of the chromosomal passenger complex (CPC), consisting of TbAUK1 and two novel proteins TbCPC1 and TbCPC2, was monitored during mitosis and cytokinesis by following the migration of the proteins tagged with enhanced yellow fluorescence protein in live cells with time-lapse video microscopy. Inhibition of TbAUK1 function in S-phase, prophase or metaphase invariably arrests the cells in the metaphase, suggesting an action of TbAUK1 in promoting metaphase-anaphase transition. TbAUK1 inhibition in anaphase does not affect mitotic exit, but prevents trans-localization of the CPC from the spindle midzone to the anterior tip of the new flagellum attachment zone for cytokinetic initiation. The CPC in the midzone is dispersed back to the two segregated nuclei, while cytokinesis is inhibited. In and beyond telophase, TbAUK1 inhibition has no effect on the progression of cytokinesis or the subsequent G1, S and G2 phases until a new metaphase is attained. There are thus two clearly distinct points of TbAUK1 action in T. brucei: the metaphase-anaphase transition and cytokinetic initiation. This is the first time to our knowledge that the dual functions of an Aurora B homolog is dissected and separated into two clearly distinct time frames in a cell cycle.

Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.

The anaerobic parasitic protozoa Tritrichomonas foetus is found incapable of de novo pyrimidine biosynthesis by its failure to incorporate bicarbonate, aspartate, or orotate into pyrimidine nucleotides or nucleic acids. Uracil phosphoribosyltransferase in the cytoplasm provides the major pyrimidine salvage for the parasite. Exogenous uridine and cytidine are mostly converted to uracil by uridine phosphorylase and cytidine deaminase in T.foetu prior to incorporation. T. foetus cannot incorporate labels from exogenous uracil or uridine into DNA; it has no detectable dihydrofolate reductase or thymidylate synthetase and is resistant to methotrexate, pyrimethamine, trimethoprim, and 5-bromovinyldeoxyuridine at millimolar concentrations. It has an enzyme thymidine phosphotransferase in cellular fraction pelleting at 100,000 x g that can convert exogenous thymidine to TMP via a phosphate donor such as p-nitrophenyl phosphate or nucleoside 5'-monophosphate. Thymidine salvage in T. foetus is thus totally dissociated from other pyrimidine salvage.It has become apparent in recent years that parasitic protozoa are generally incapable of de novo synthesis of purine nucleotides. Trypanosoma cruzi (1), Leishmania donovani (2), Plasmodium lophurae (3), Eimeria tenella (4), and Trichomonas vaginalis (5), to name but a few examples, depend on specific networks of salvage pathways to fulfill their purine requirements. Because of this deficiency in metabolic activities, rational approaches to controlling some of the parasites have been possible. Allopurinol exhibits antitrypanosomal and antileishmanial activities because it is recognized by the parasite salvage enzymes as a hypoxanthine analog (6, 7). Allopurinol riboside (8), formycin B (9), and 4-thiopyrazolopyrimidine riboside (10) have antileishmanial activities because of the nucleoside phosphotransferase in leishmania, which incorporates the compounds into parasite's nucleotide pool.De novo pyrimidine biosynthesis, on the other hand, takes place in most of the parasitic protozoa. Recently, it has been reported that the anaerobic flagellates Trichomonas vaginalis and Giardia lamblia may not, however, have even the capability of pyrimidine de novo synthesis. The former lacks aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, and orotate phosphoribosyltransferase in its crude extract (11), whereas the latter indicates no incorporation of aspartate into the cold trichloroacetic acid-insoluble fraction (12). These results suggest that anaerobic flagellates may differ from other protozoan parasites in lacking both purine and pyrimidine de novo synthetic abilities and thus may offer even more opportunities for chemotherapeutic attack.To verify these possibilities, we studied pyrimidine metabolism in Tritrichomonasfoetus, a Precursor Incorporation into the Nucleotide Pool. T. foetus cells were washed, suspended in phosphate-buffered saline, pH 7.2 (Pi/NaCl)/20 mM glucose to a final cell density of 108/ml, and incubated at 370C. A radiolabeled subst...