BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth of Trypanosoma brucei.

RNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 in Trypanosoma brucei (TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics. In silico analysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of five α-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms of T. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

Identification of protein complex associated with LYT1 of Trypanosoma cruzi.

To carry out the intracellular phase of its life cycle, Trypanosoma cruzi must infect a host cell. Although a few molecules have been reported to participate in this process, one known protein is LYT1, which promotes lysis under acidic conditions and is involved in parasite infection and development. Alternative transcripts from a single LYT1 gene generate two proteins with differential functions and compartmentalization. Single-gene products targeted to more than one location can interact with disparate proteins that might affect their function and targeting properties. The aim of this work was to study the LYT1 interaction map using coimmunoprecipitation assays with transgenic parasites expressing LYT1 products fused to GFP. We detected several proteins of sizes from 8 to 150 kDa that bind to LYT1 with different binding strengths. By MS-MS analysis, we identified proteins involved in parasite infectivity (trans-sialidase), development (kDSPs and histones H2A and H2B), and motility and protein traffic (dynein and α - and ß -tubulin), as well as protein-protein interactions (TPR-protein and kDSPs) and several hypothetical proteins. Our approach led us to identify the LYT1 interaction profile, thereby providing insights into the molecular mechanisms that contribute to parasite stage development and pathogenesis of T. cruzi infection.

Flagellar motility of Trypanosoma cruzi epimastigotes.

The hemoflagellate Trypanosoma cruzi is the causative agent of American trypanosomiasis. Despite the importance of motility in the parasite life cycle, little is known about T. cruzi motility, and there is no quantitative description of its flagellar beating. Using video microscopy and quantitative vectorial analysis of epimastigote trajectories, we find a forward parasite motility defined by tip-to-base symmetrical flagellar beats. This motion is occasionally interrupted by base-to-tip highly asymmetric beats, which represent the ciliary beat of trypanosomatid flagella. The switch between flagellar and ciliary beating facilitates the parasite's reorientation, which produces a large variability of movement and trajectories that results in different distance ranges traveled by the cells. An analysis of the distance, speed, and rotational angle indicates that epimastigote movement is not completely random, and the phenomenon is highly dependent on the parasite behavior and is characterized by directed and tumbling parasite motion as well as their combination, resulting in the alternation of rectilinear and intricate motility paths.

Improved method for in vitro secondary amastigogenesis of Trypanosoma cruzi: morphometrical and molecular analysis of intermediate developmental forms.

Trypanosoma cruzi undergoes a biphasic life cycle that consists of four alternate developmental stages. In vitro conditions to obtain a synchronic transformation and efficient rates of pure intermediate forms (IFs), which are indispensable for further biochemical, biological, and molecular studies, have not been reported. In the present study, we established an improved method to obtain IFs from secondary amastigogenesis. During the transformation kinetics, we observed progressive decreases in the size of the parasite body, undulating membrane and flagellum that were concomitant with nucleus remodeling and kinetoplast displacement. In addition, a gradual reduction in parasite movement and acquisition of the amastigote-specific Ssp4 antigen were observed. Therefore, our results showed that the in vitro conditions used obtained large quantities of highly synchronous and pure IFs that were clearly distinguished by morphometrical and molecular analyses. Obtaining these IFs represents the first step towards an understanding of the molecular mechanisms involved in amastigogenesis.

Genomic organization and functional characterization of the Leishmania major Friedlin ribosomal RNA gene locus.

The sequence and gene organization of the ribosomal RNA (rRNA) genes of Leishmania major Friedlin (LmjF) were determined. Interestingly, the rDNA repeat unit contained a duplicated 526 bp fragment at the 3' end of the unit with two copies of the LSUepsilon rRNA gene. Our results suggested the presence of only approximately 24 copies of the rRNA unit per diploid genome in LmjF. Repetitive elements (IGSRE) of 63 bp occurred in the intergenic spacer (IGS) between the LSUepsilon and the SSU rRNA genes. Among the different rDNA units, the region containing the IGSRE fluctuated in length from approximately 1.3 to approximately 18 kb. The transcription initiation site (TIS) of the rRNA unit was localized by primer extension to 1043 bp upstream of the SSU gene and 184 bp downstream of the IGSRE. Sequence comparison among several species of Leishmania showed a high degree of conservation around the TIS. Moreover, the IGSRE also showed considerable similarity between Leishmania species. In transient transfection assays, a fragment containing the TIS directed a 164- to 178-fold increase in luciferase activity over the no-insert control, indicating the presence of a promoter within this 391 bp fragment. The LmjF promoter region was also functional in other species of Leishmania. Nuclear run-on analyses demonstrated that only the rRNA-coding strand is transcribed, downstream of this RNA polymerase I (pol I) promoter. These experiments also suggested that transcription terminates upstream of the IGSRE.

The Leishmania genome project: new insights into gene organization and function.

The sequencing of Leishmania major Friedlin chromosome 1 (Chr1), Chr3, and Chr4 has been completed. and several other chromosomes are well underway. The complete genome sequence should be available by 2003. Over 1,000 full-length new genes have been identified, with the majority (approximately 75%) having unknown function. Many of these may be Leishmania (or kinetoplastid) specific. Most interestingly, the genes are organized into large (> 100-500 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a "divergent" manner, i.e., the mRNAs for the two sets of genes are both transcribed towards the telomeres. Nuclear run-on analysis suggests that transcription is initiated in both directions within the "divergent" region. Chr3 and Chr4 contain two "convergent" clusters, with a single "divergent" gene at one telomere of Chr3. Sequence analysis of several genes from the LD1 region of Chr35 indicates a high degree of sequence conservation between L. major and L. donovani/L. infantum within protein-coding open reading frames (ORFs), with a lower degree of conservation within the non-coding regions. Immunization of mice with recombinant antigen from two of these genes, BTI (formerly ORFG) and ORFF, results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

Genomic organization and gene function in Leishmania.

Sequencing of the Leishmania major Friedlin genome is well underway with chromosome 1 (Chr1) and Chr3 having been completely sequenced, and Chr4 virtually complete. Sequencing of several other chromosomes is in progress and the complete genome sequence may be available as soon as 2003. A large proportion ( approximately 70%) of the newly identified genes remains unclassified, with many of these being potentially Leishmania- (or kinetoplastid-) specific. Most interestingly, the genes are organized into large (>100-300 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a 'divergent' manner, i. e. the mRNAs for the two sets of genes are both transcribed towards the telomeres. Chr3 contains two 'convergent' clusters, with a single 'divergent' gene at one telomere, with the two large clusters separated by a tRNA gene. We have characterized several genes from the LD1 (Leishmania DNA 1) region of Chr35. BT1 (formerly ORFG) encodes a biopterin transporter and ORFF encodes a nuclear protein of unknown function. Immunization of mice with recombinant antigens from these genes results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

The deduced primary structure of a ribosomal protein S4 from Trypanosoma cruzi.

Two cDNA clones encoding a protein homologous to ribosomal protein S4 from the protozoan parasite Trypanosoma cruzi were isolated and characterized. Both clones potentially encode for an identical basic protein of 273 amino acids. Sequence comparisons with other species indicate that this protein is highly conserved. Hybridization studies are consistent with the occurrence of two genes in T. cruzi encoding this ribosomal protein. An RNA of approximately 1 kb was present in both exponentially growing and stationary phase derived epimastigotes, and no significant differences were detected in its steady state concentration.

pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants.

To improve the selection phenotype of the expression plasmid pTEX, a Trypanosoma cruzi rDNA (DNA coding for rRNA) gene spacer fragment (806 bp) containing a mapped transcription start point (tsp) was cloned in the vectors pTEX and pTEX-cat, generating the plasmids pRIBOTEX and pRIBOTEX-cat. T. cruzi cultures transiently transfected with pRIBOTEX-cat expressed a chloramphenicol (Cm) acetyltransferase (CAT) activity 16,000-fold greater than the activity observed with the parental vector pTEX-cat. Moreover, T. cruzi cells transformed with pRIBOTEX and pRIBOTEX-cat exhibited logarithmic growth in the presence of Geneticin (G418) 2 weeks earlier than that observed with controls transformed with pTEX. The plasmid copy number in stably transformed trypanosomes was about 50-times higher in cultures transformed with pTEX-cat than in cells transformed with pRIBOTEX or pRIBOTEX-cat. However, the neo RNA steady-state level and the CAT activity observed among the stably transfected cultures showed only modest differences. Finally, it was found that the pRIBOTEX vector was not episomally maintained as pTEX, but integrated into a chromosome indistinguishable from the one encoding rRNA. These features make pRIBOTEX a useful tool for transfection and rapid expression of genes in T. cruzi.

Trypanosoma cruzi rRNA genes: a repeated element from the non-transcribed spacer is locus specific.

To determine the occurrence of conserved domains of presumed functional selection, a genomic restriction analysis was carried out in the region surrounding a transcription start point (tsp) from the rRNA cistron in T. cruzi. The transcribed spacer was found highly conserved among several isolates, whereas at 146 bp upstream from the tsp a highly polymorphic pattern was evidenced with a probe that contains sequences of a repetitive element (172 bp). Both genomic and chromosomal hybridizations indicated the linkage of the repetitive element to coding regions of the rRNA cistron. This represents the first example of a repetitive element not interspersed throughout the genome of T. cruzi, and strongly suggests that a functional role is being selected.

Trypanosoma cruzi ribosomal DNA: mapping of a putative distal promoter.

The nucleotide (nt) sequence (2106 bp) of a cloned rDNA (encoding ribosomal RNA) spacer region from Trypanosoma cruzi was determined and a putative transcription start point (tsp) was mapped. The assigned length for the transcribed spacer is 1768 bp and its tsp is present 270-bp upstream from an alternative tsp published for the equivalent gene from another T. cruzi strain [Dietrich et al., Gene 125 (1993) 103-107]. Sequence comparisons of the nt flanking both T. cruzi tsp with the homologous regions from both other trypanosomatids, and other eukaryotes, indicate that these sequences are poorly conserved within the family Trypanosomatidae. This finding reinforces the proposal that the speciation of trypanosomatids may have occurred early in evolution.

Trypanosoma cruzi ribosomal RNA genes: a review.

Our research work group has been interested in the study of the ribosomal RNA and 5S gene systems from Trypanosoma cruzi. Our contributions span from the general description of a multifragmented molecular system, to the sequence analysis of some ribosomal RNA coding regions. From the latter, we have constructed phylogenetic trees of the Trypanosomatidae family, and our data indicate that the molecular inferences do not sustain the traditional classification of these species. Our published findings are here reviewed along with recent unpublished observations of ribosomal RNA and 5S gene structures.

Trypanosoma cruzi 5S rRNA genes: molecular cloning, structure and chromosomal organization.

To further study the ribosomal RNA genetic system in Trypanosoma cruzi, the 5S rRNA gene family was characterized. We found that this gene family is reiterated about 1600 times per diploid nuclei and is mostly organized as a tandem repeat of 481 base pairs. These gene clusters were assigned to two chromosomes of about 1500 and 1400 kilobase pairs. We found that the 5S rRNA-coding region is comprised of 120 nucleotides, and contains the well-known internal control regions of eukaryotic RNA polymerase III. The two gene-spacer regions analysed exhibit a putative signal for transcription termination and six sites homologous to the consensus sequence for the binding of transcription factor Sp1.