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Trypanosomatid protozoa are unusual eukaryotes that are well known for having unusual ways of controlling their gene expression. The lack of a refined mode of transcriptional control in these organisms is compensated by several post-transcriptional control mechanisms, such as control of mRNA turnover and selection of mRNA for translation, that may modulate protein synthesis in response to several environmental conditions found in different hosts. In other eukaryotes, selection of mRNA for translation is mediated by the complex eIF4F, a heterotrimeric protein complex composed by the subunits eIF4E, eIF4G, and eIF4A, where the eIF4E binds to the 5′-cap structure of mature mRNAs. In this review, we present and discuss the characteristics of six trypanosomatid eIF4E homologs and their associated proteins that form multiple eIF4F complexes. The existence of multiple eIF4F complexes in trypanosomatids evokes exquisite mechanisms for differential mRNA recognition for translation.

Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.

Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

The protozoan Trypanosoma cruzi, its mammalian reservoirs, and vectors have existed in nature for millions of years. The human infection, named Chagas disease, is a major public health problem for Latin America. T. cruzi is genetically highly diverse and the understanding of the population structure of this parasite is critical because of the links to transmission cycles and disease. At present, T. cruzi is partitioned into six discrete typing units (DTUs), TcI-TcVI. Here we focus on the current status of taxonomy-related areas such as population structure, phylogeographical and eco-epidemiological features, and the correlation of DTU with natural and experimental infection. We also summarize methods for DTU genotyping, available for widespread use in endemic areas. For the immediate future multilocus sequence typing is likely to be the gold standard for population studies. We conclude that greater advances in our knowledge on pathogenic and epidemiological features of these parasites are expected in the coming decade through the comparative analysis of the genomes from isolates of various DTUs.

The 5' cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5'-5' triphosphate bond followed by 2'-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2'-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N(7) methylation of the guanosine cap nor 2'-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2'-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

To develop molecular markers for lower trypanosmatids, we have examined the mini-exon gene repeats of 17 isolates that were classified as Crithidia by traditional methods. Representative repeats were amplified by polymerase chain reaction and the amplification products were cloned and used as hybridization probes against genomic DNA. Six hybridization groups of Crithidia were defined on the basis of the DNA blotting experiments. The three endosymbiont-bearing species (C. deanei, C. desouzai and C. oncopelti) and C. acanthocephali each belonged to single-member hybridization groups, while the C. fasciculata group contained additional named and undesignated species. The Crithidia lucilae thermophila probe hybridized to multiple undesignated isolates. The DNA sequence of the cloned products revealed that the specificity of the hybridization probes was due to substantial differences in the intron and the non-transcribed spacer regions. These data indicate substantial heterogeneity within the mini-exon gene locus of the taxon Crithidia.

In kinetoplastids a 39-nucleotide spliced leader RNA is trans-spliced to the 5′ end of nuclear mRNAs before they can be translated, thus the spliced leader is central to gene expression in kinetoplastid biology. The spliced leader RNA genes in Leishmania tarentolae contain promoters with important sites at approximately −60 and −30. A complex forms specifically on the −60 element as shown by electrophoretic mobility shift. The −60 shift complex has an estimated mass of 159 kDa. An L. tarentolae homologue of TATA-binding protein, Lt TBP, co-fractionates with the −60 shift complex. Inclusion of anti- Lt TBP antiserum in the shift assay disrupts the shift, indicating that Lt TBP is a component of the complex that interacts with the TATA-less −60 element of the spliced leader RNA gene promoter. Both Lt TBP and Lt SNAP 50 are found near the spliced leader RNA gene promoter and the promoters important for tRNA Ala and/or U2 snRNA gene transcription, as demonstrated by chromatin immunoprecipitation. The Lt TBP appears to interact with a subset of promoters in kinetoplastids with an affinity for short transcription units.

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3' extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 3'-->5' exonuclease required for SL RNA 3'-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3' processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3'-->5' proofreading epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3' extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3' extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3'-end formation is a multistep process in which SNIP provides the ultimate 3'-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.

As a consequence of infection by Trypanosoma cruzi, 30% of victims may develop chronic Chagas disease, which presents a spectrum of pathology including cardiomyopathy, megacolon and megaesophagus. The outcome of infection in a particular individual is the result of a set of complex interactions among the host genetic background, environmental and social factors, and the genetic composition of the parasite, all of which can be complicated by mixed infections and re-infections. Initially we consider what is known about the genetic structure and biological properties of the protozoan. Currently, six distinct subgroups have been characterized by different combinations of four distinct genotypic classes. The recent demonstration of genetic exchange via non-meiotic cell fusion illustrates a mechanism by which maintained heterogeneous polyploidy may have been generated in these parasites. Subsequently, we consider factors in humans and in experimental mouse-infection and tissue culture models that have contributed to our understanding of the host's susceptibility or resistance to disease. Identification of the direct players in host-pathogen interactions at the establishment and chronic phases of the disease is perhaps the best hope of a clinical handle for treatment. At some point in the future, these disparate areas of study will have to come together. It is to be hoped that this scientific fusion will result in better prognosis and treatment of Chagas disease.

The Sm-binding site of the kinetoplastid spliced leader RNA has been implicated in accurate spliced leader RNA maturation and trans -splicing competence. In Trypanosoma brucei , RNA interference-mediated knockdown of SmD1 caused defects in spliced leader RNA maturation, displaying aberrant 3′-end formation, partial formation of cap 4, and overaccumulation in the cytoplasm; U 28 pseudouridylation was unaffected.

A role for parasite genetic variability in the spectrum of Chagas disease is emerging but not yet evident, in part due to an incomplete understanding of the population structure of Trypanosoma cruzi. To investigate further the observed genotypic variation at the sequence and chromosomal levels in strains of standard and field-isolated T. cruzi we have undertaken a comparative analysis of 10 regions of the genome from two isolates representing T. cruzi I (Dm28c and Silvio X10) and two from T. cruzi II (CL Brener and Esmeraldo). Amplified regions contained intergenic (non-coding) sequences from tandemly repeated genes. Multiple nucleotide polymorphisms correlated with the T. cruzi I/T. cruzi II classification. Two intergenic regions had useful polymorphisms for the design of classification probes to test on genomic DNA from other known isolates. Two adjacent nucleotide polymorphisms in HSP 60 correlated with the T. cruzi I and T. cruzi II distinction. 1F8 nucleotide polymorphisms revealed multiple subdivisions of T. cruzi II: subgroups IIa and IIc displayed the T. cruzi I pattern; subgroups IId and IIe possessed both the I and II patterns. Furthermore, isolates from subgroups IId and IIe contained the 1F8 polymorphic markers on different chromosome bands supporting a genetic exchange event that resulted in chromosomes V and IX of T. cruzi strain CL Brener. Based on these analyses, T. cruzi I and subgroup IIb appear to be pure lines, while subgroups IIa/IIc and IId/IIe are hybrid lines. These data demonstrate for the first time that IIa/IIc are hybrid, consistent with the hypothesis that genetic recombination has occurred more than once within the T. cruzi lines.

Debora M. Santana a,b, Gulnara P. Borja-Cabrera b, Edilma Paraguai de Souza b, Nancy R. Sturm a, Clarisa B. Palatnik de Sousa b, David A. Campbell * a Department of Microbiology, Immunology and Molecular Genetics, School of Medicine, University of California, 10833 Le Conte Avenue, Los Angeles, CA 90095-1747, USA b Instituto de Microbiologia*/Professor Paulo de Goes, CCS, Cidade Universitaria, Ilha do Fundao, UFRJ, CP 68040, CEP 21944-590 Rio de Janeiro, RJ, Brazil

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., Author Summary Some plant trypanosomes, single-celled organisms living in phloem sap, are responsible for important palm diseases, inducing frequent expensive and toxic insecticide treatments against their insect vectors. Other trypanosomes multiply in latex tubes without detriment to their host. Despite the wide range of behaviors and impacts, these trypanosomes have been rather unceremoniously lumped into a single genus: Phytomonas. A battery of molecular probes has been used for their characterization but no clear phylogeny or classification has been established. We have sequenced the genomes of a pathogenic phloem-specific Phytomonas from a diseased South American coconut palm and a latex-specific isolate collected from an apparently healthy wild euphorb in the south of France. Upon comparison with each other and with human pathogenic trypanosomes, both Phytomonas revealed distinctive compact genomes, consisting essentially of single-copy genes, with the vast majority of genes shared by both isolates irrespective of their effect on the host. A strong cohort of enzymes in the sugar metabolism pathways was consistent with the nutritional environments found in plants. The genetic nuances may reveal the basis for the behavioral differences between these two unique plant parasites, and indicate the direction of our future studies in search of effective treatment of the crop disease parasites.

We report the cloning and sequencing of the first minicircle from a phloem-restricted, pathogenic Phytomonas sp. (Hart 1) isolated from a coconut palm with hartrot disease. The minicircle possessed a two-domain structure of two conserved regions, each containing three conserved sequence blocks (CSB). Based on the sequence around CSB 3 from Hart 1, PCR primers were designed to allow specific amplification of Phytomonas minicircles. This primer pair demonstrated specificity for at least six groups of plant trypanosomatids and did not amplify from insect trypanosomatids. The PCR results were consistent with a two-domain structure for other plant trypanosomatids.

The spliced leader RNA genes of Bodo saltans, Cryptobia helicis and Dimastigella trypaniformis were analyzed as molecular markers for additional taxa within the suborder Bodonina. The non-transcribed spacer regions were distinctive for each organism, and 5S rRNA genes were present in Bodo and Dimastigella but not in C. helicis. Two sequence classes of 5S rRNA were evident from analysis of the bodonid genes. The two classes of 5S rRNA genes were found in other Kinetoplastids independent of co-localization with the spliced leader RNA gene.

To elucidate the process of transcription in the kinetoplastids and to aid in the purification of transcription factors, we have developed a transcriptionally-competent nuclear extract from Leishmania tarentolae for the study of the spliced leader (SL) RNA gene. The extract was competent to transcribe a tagged SL RNA gene. The in vitro SL RNA transcripts initiated accurately and their synthesis was dependent on the presence of the promoter defined in vivo. The nuclear extract was then challenged rigorously using an exhaustive set of mutated SL RNA gene templates previously tested for transcriptional activity in vivo. Mutation of four nucleotides (CCGG) at positions -34 to -31 had a detrimental effect on transcription in vitro: the CC dinucleotide overlaps one element necessary in vivo. Similarly. four nucleotides (TGAC; positions -67 to -64) important for transcription in vitro overlapped the other core promoter element defined in vivo, but were generally not effective as point mutations. The promoter-binding ability of the transcriptionally-competent extract for the -60 region mutations mirrored the in vitro transcription pattern. Although it does not reflect precisely the in vivo results, this in vitro system provides us with an important tool for monitoring the purification of potential transcription factors, as well as the basis for future reconstitution experiments.

A 39-nucleotide leader is trans-spliced onto all trypanosome nuclear mRNAs. The precursor spliced leader RNA was tested for trans-splicing function in vivo by mutating the intron. We report that in Leishmania tarentolae spliced leader RNA 5' modification is influenced by the primary sequence of stem-loop II, the Sm-binding site, and the secondary structure of stem-loop III. The sequence of stem-loop II was found to be important for cap 4 formation and splicing. As in Ascaris, mutagenesis of the bulge nucleotide in stem-loop II was detrimental to trans-splicing. Because restoration of the L. tarentolae stem-loop II structure was not sufficient to restore splicing, this result contrasts the findings in the kinetoplastid Leptomonas, where mutations that restored stem-loop II structure supported splicing. Methylation of the cap 4 structure and splicing was also dependent on both the Sm-binding site and the structure of stem-loop III and was inhibited by incomplete 3' end processing. The critical nature of the L. tarentolae Sm-binding site is consistent with its essential role in the Ascaris spliced leader RNA, whereas in Leptomonas mutation of the Sm-binding site and deletion of stem-loop III did not affect trans-splicing. A pathway for Leishmania spliced leader RNA processing and maturation is proposed.

The mini-exon gene repeats from two isolates of Trypanosoma (Nannomonas) simiae were amplified by polymerase chain reaction (PCR). The products from each isolate differed in size, however they cross-hybridised in Southern blots. The nature of the size variation was revealed by comparison of the DNA sequence of each repeat: relative to the BAN7 strain, the mini-exon gene of KETRI 2431 contained three apparent deletions that were flanked by short (or = 6-bp) direct repeats. Furthermore, one of the cloned repeats was used as a hybridisation probe against DNA from other closely-related African trypanosomes. The lack of hybridisation of the T. (N.) simiae mini-exon gene to genomic DNA from the Forest, Kilifi and Savannah subgroups of T. (N.) congolense and T. (N.) godfreyi indicate that this PCR-hybridisation assay may be useful for distinguishing T. (N.) simiae from other members of the subgenus Nannomonas.

Posttranscriptional modifications to the rRNAs, including endonucleolytic cleavage, 2′-O-ribose methylation and pseudouridinylation, are mediated by small, nucleolar RNAs (snoRNAs). The most abundant snoRNA, U3, has been identified in numerous eukaryotes, including the kinetoplastids (27) and Euglena sp. (23). A requirement for U3 snoRNA in endonucleolytic cleavages of precursor rRNA transcripts has been established in yeast and vertebrate systems (31, 36). Multiple other snoRNAs have been identified that are involved in the 2′-O-ribose methylation and pseudouridinylation of the 18S and 25/28S rRNAs (3, 22, 42, 43), although the precise function of many snoRNAs remains unclear. Several criteria have been used to identify snoRNAs (24, 36, 50), including the presence of conserved sequence motifs, association with nucleolar proteins (fibrillarin, Gar1p, and Pop1p), complementarity to pre-rRNAs, and localization to the nucleolus. Like other cellular RNAs, snoRNAs are believed to exist as ribonucleoprotein complexes (36, 50) and have been divided into two major classes based on conserved sequence elements and protein associations. The box C/D snoRNAs usually contain box C and box D elements near their 5′ and 3′ ends, respectively; these elements are required for snoRNP interaction with the abundant nucleolar protein, fibrillarin. In vertebrates, box C/D snoRNAs are frequently processed from the introns of protein-encoding mRNAs (54), while in plants and yeasts they may be transcribed polycistronically (32, 50). The other class, the box H/ACA snoRNAs, contains two conserved sequence motifs: box H resides in a hinge region between two stem-loop structures, and the “ACA” trinucleotide motif resides 3 nucleotides (nt) from their 3′ ends (3, 22). Box H/ACA snoRNAs of yeasts associate with the essential Gar1 protein (22). Most members of each class of snoRNAs function in modification of rRNA sequences: box C/D snoRNAs guide 2′-O-ribose methylation, and box H/ACA snoRNAs are involved in pseudouridinylation (21). Box C/D snoRNAs direct modification of rRNAs through stretches of sequence complementarity. The region of base pairing between box C/D snoRNAs and the site of methylation in the rRNA always lies immediately upstream from a D box or a D′ box, which is a slightly degenerate version of the D box (29, 43, 55). A number of other snoRNAs containing box C and D elements (U3, U8, U14, and U22) or box H/ACA elements (for example, U17/E1 [19] and snR30 [42]) do not appear to play roles in rRNA nucleotide modification, but they may be required for pre-rRNA cleavages or have roles in pre-RNA folding and ribosome assembly (50). A number of small nuclear RNAs have been described in the kinetoplastids: the spliced leader (SL) RNA (12, 30, 38), four U small nuclear RNAs involved in trans splicing (U2, U4, and U6 [39, 51, 52] and U5 [18, 60]), the SL-associated (SLA) RNA (44, 46, 58), the mitochondrial guide RNAs (6, 49), the 7SL RNAs (37), the tRNAs (11, 40), the six large-subunit (LSU) rRNA fragments (15, 28), and the U3 snoRNA (27, 39). The U3 RNA is the only snoRNA identified in this group of organisms. We have characterized four small RNAs in Leishmania tarentolae and Trypanosoma cruzi that are similar to those originally identified as transcripts of unknown function from the SLA RNA repeat in T. brucei (46). We present evidence that three transcripts fractionate with nucleolar markers. Two of the transcripts are bona fide snoRNAs: the 75/76- and 92/94-nt RNAs contain box C, D, and D′ elements and 10- to 14-bp stretches with complementarity to sites in the small-subunit (SSU) or LSU rRNAs. We show that one nucleotide within each of these blocks is ribose methylated. The positions of methylated nucleotides relative to the D or D′ box in the complementary snoRNA differ from the canonical 5-bp spacing seen in higher eukaryotes; in the two sites described here the methylated ribose is the first in the stretch of complementarity. This suggests that the 5-bp rule that directs rRNA methylation in the higher eukaryotes may not be conserved among the early-diverging kinetoplastids.

In dinoflagellates and perkinsids, the molecular structure of the protein translocating machinery is unclear. Here, we identified several types of full-length signal recognition particle (SRP) RNA genes from Karenia brevis (dinoflagellate) and Perkinsus marinus (perkinsid). We also identified the four SRP S-domain proteins, but not the two Alu domain proteins, from P. marinus and several dinoflagellates. We mapped both ends of SRP RNA transcripts from K. brevis and P. marinus, and obtained the 3′ end from four other dinoflagellates. The lengths of SRP RNA are predicted to be ∼260-300 nt in dinoflagellates and 280-285 nt in P. marinus. Although these SRP RNA sequences are substantially variable, the predicted structures are similar. The genomic organization of the SRP RNA gene differs among species. In K. brevis, this gene is located downstream of the spliced leader (SL) RNA, either as SL RNA-SRP RNA-tRNA gene tandem repeats, or within a SL RNA-SRP RNA-tRNA-U6-5S rRNA gene cluster. In other dinoflagellates, SRP RNA does not cluster with SL RNA or 5S rRNA genes. The majority of P. marinus SRP RNA genes array as tandem repeats without the above-mentioned small RNA genes. Our results capture a snapshot of a potentially complex evolutionary history of SRP RNA in alveolates.

FUD6, a nonphotosynthetic mutant of Chlamydomonas reinhardtii, was previously found to be deficient in the synthesis of subunit IV of the cytochrome b6/f complex, the chloroplast petD gene product (C. Lemaire, J. Girard-Bascou, F.-A. Wollman, and P. Bennoun, Biochim. Biophys. Acta 851:229-238, 1986). The lesion in FUD6 is a 236-bp deletion between two 11-bp direct repeats in the chloroplast genome. It extends from 82 to 72 bp upstream of the 5' end of wild-type petD mRNA to 156 to 166 bp downstream of the 5' end. Thus, the deletion extends into the putative promoter and 5' untranslated region of petD. No petD mRNA of the normal size can be detected in FUD6 cells, but a low level of a dicistronic message accumulates, which contains the coding regions for subunit IV and cytochrome f, the product of the upstream petA gene. petD transcriptional activity in FUD6 is not significantly altered from the wild-type level. This transcriptional activity was eliminated by petA promoter disruptions, suggesting that it originates at the petA promoter. We conclude that the petD-coding portion of most cotranscripts is rapidly degraded in FUD6, possibly following processing events that generate the 3' end of petA mRNA. A chloroplast transformant was constructed in which only the sequence from -81 to -2 relative to the major 5' end of the petD transcript was deleted. Although this deletion eliminates all detectable petD promoter activity, the transformant grows phototrophically and accumulates high levels of monocistronic petD mRNA. We conclude that the petD gene can be transcribed by functionally redundant promoters. In the absence of a functional petD promoter, a lack of transcription termination allows the downstream petD gene to be cotranscribed with the petA coding region and thereby expressed efficiently.

The distinction between plant trypanosomatids and opportunistic monoxenous insect trypanosomatids has not been demarcated clearly due to the mass placement of all trypanosomatids isolated from plants into the arbitrary genus Phytomonas spp. The advent of molecular markers has been useful in distinguishing plant trypanosomatids from the rest of the Trypanosomatidae family. Here we have examined the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) locus for classification purposes. This region contains two distinct ITSs flanked by the small subunit and large subunit of ribosomal RNA genes and separated by the 5.8S ribosomal RNA gene. Sequences within the 5.8S ribosomal RNA gene and in the ITS sequences can serve as specific markers for several of the Phytomonas groups. Microsatellite sequences were identified in Phytomonas spp. in both ITS regions. Several classes of microsatellites were seen, with inter-isolate variation that has potential for future use. Maximum Likelihood analysis of the ITS sequences of 20 Phytomonas isolates representing the eight defined groups and a few unclassified isolates revealed a total of 10 distinct subgroups within our collection, of which two are new. The ITS region, which includes the 5.8S sequence, is a robust marker for the subdivisions within the genus Phytomonas spp.

Through trans-splicing of a 39-nt spliced leader (SL) onto each protein-coding transcript, mature kinetoplastid mRNA acquire a hypermethylated 5'-cap structure, but its function has been unclear. Gene deletions for three Trypanosoma brucei cap 2'-O-ribose methyltransferases, TbMTr1, TbMTr2 and TbMTr3, reveal distinct roles for four 2'-O-methylated nucleotides. Elimination of individual gene pairs yields viable cells; however, attempts at double knock-outs resulted in the generation of a TbMTr2-/-/TbMTr3-/- cell line only. Absence of both kinetoplastid-specific enzymes in TbMTr2-/-/TbMTr3-/- lines yielded substrate SL RNA and mRNA with cap 1. TbMTr1-/- translation is comparable with wildtype, while cap 3 and cap 4 loss reduced translation rates, exacerbated by the additional loss of cap 2. TbMTr1-/- and TbMTr2-/-/TbMTr3-/- lines grow to lower densities under normal culture conditions relative to wildtype cells, with growth rate differences apparent under low serum conditions. Cell viability may not tolerate delays at both the nucleolar Sm-independent and nucleoplasmic Sm-dependent stages of SL RNA maturation combined with reduced rates of translation. A minimal level of mRNA cap ribose methylation is essential for trypanosome viability, providing the first functional role for the cap 4.

Spliced leader (SL) trans-splicing is a common mRNA processing mechanism in dinoflagellates, in which a 22-nt sequence is transferred from the 5′-end of a small noncoding RNA, the SL RNA, to the 5′-end of mRNA molecules. Although the SL RNA gene was shown initially to be organized as tandem repeats with transcripts of 50–60 nt, shorter than most of their counterparts in other organisms, other gene organizations and transcript lengths were reported subsequently. To address the evolutionary gradient of gene organization complexity, we thoroughly examined transcript and gene organization of the SL RNA in a phylogenetically and ecologically diverse group of dinoflagellates representing four Orders. All these dinoflagellates possessed SL RNA transcripts of 50–60 nt, although in one species additional transcripts of up to 92 nt were also detected. At the genomic level, various combinations of SL RNA and 5S rRNA tandem gene arrays, including SL RNA–only, 5S rRNA–only, and mixed SL RNA–5S rRNA (SL–5S) clusters, were amplified by polymerase chain reaction for six dinoflagellates, containing intergenic spacers ranging from 88 bp to over 1.2 kb. Of these species, no SL–5S cluster was detected in Prorocentrum minimum, and only Karenia brevis showed the U6 small nuclear RNA gene associated with these mixed arrays. The 5S rRNA–only array was also found in three dinoflagellates, along with two SL–5S-adjacent arrangements found in two other species that could represent junctions. Two species contained multimeric SL exon repeats with no associated intron. These results suggest that 1) both the SL RNA tandem repeat and the SL–5S cluster genomic organizations are an “ancient” and widespread feature within the phylum of dinoflagellates and 2) rampant genomic duplication and recombination are ongoing independently in each dinoflagellate lineage, giving rise to the highly complex and diversified genomic arrangements of the SL RNA gene, while conserving the length and structure of the functional SL RNA.

Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2-O-ribose methylation of the first four nucleotides and pseudouridine () formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL 28 formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3-end maturation, and 28 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate. Mature RNA molecules contain extensive posttranscriptional nucleotide modifications of both base and ribose moieties. The isomerization of uracil to pseudouridine (5-ribosyluracil) and 2O-ribose methylation occurs by either site-specific (31, 50) or small nucleolar RNA (snoRNA)-guided enzymes (13). Conserved snoRNA-guided rRNA nucleotide modifications occur in functionally relevant regions (14), with their absence resulting in disease states due to defective catalytic RNA activity (76). Cap ribose methylations of RNA polymerase II transcripts by capspecific methyltransferases (MTases) are conserved in higher eukaryotes and implicated in the enhancement of translational efficiency (28a).

The completion of the genome sequencing projects for major pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania major has enabled numerous studies that would have been difficult or impossible to perform otherwise. New technologies in sequencing and protein analyses promise further rapid expansion in our capabilities. The keys to successful use of these new tools are recognizing the power and limitations of studies performed thus far, grasping the unrealized potential of new and developing technologies, and creating access to a multidisciplinary set of skills that will facilitate research, particularly in the bioinformatic analysis of the reams of data that will be forthcoming. In this Discussion, we will provide an overview of kinetoplastid genomics studies with emphasis on studies advanced through genomic data, and a preview of what may come in the near future.

In metazoa cap 1 (m(7)GpppNmp-RNA) is linked to higher levels of translation; however, the enzyme responsible remains unidentified. We have validated the first eukaryotic encoded cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family that modifies the first transcribed nucleotide of spliced leader and U1 small nuclear RNAs in the kinetoplastid protozoan Trypanosoma brucei. In addition to cap 0 (m(7)GpppNp-RNA), mRNA in these parasites has ribose methylations on the first four nucleotides with base methylations on the first and fourth (m(7)Gpppm(6,6)AmpAmpCmpm(3)Ump-SL RNA) conveyed via trans-splicing of a universal spliced leader. The function of this cap 4 is unclear. Spliced leader is the majority RNA polymerase II transcript; the RNA polymerase III-transcribed U1 small nuclear RNA has the same first four nucleotides as spliced leader, but it receives an m(2,2,7)G cap with hypermethylation at position one only (m(2,2,7)Gpppm(6,6)AmpApCpUp-U1 snRNA). Here we examine the biochemical properties of recombinant TbMTr1. Active over a pH range of 6.0 to 9.5, TbMTr1 is sensitive to Mg(2+). Positions Lys(95)-Asp(204)-Lys(259)-Glu(285) constitute the conserved catalytic core. A guanosine cap on RNA independent of its N(7) methylation status is required for substrate recognition, but an m(2,2,7G)-cap is not recognized. TbMTr1 favors the spliced leader 5' sequence, as reflected by a preference for A at position 1 and modulation of activity for substrates with base changes at positions 2 and 3. With similarities to human cap 1 methyltransferase activity, TbMTr1 is an excellent model for higher eukaryotic cap 1 methyltransferases and the consequences of cap 1 modification.

mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.

Background The spliced leader (SL) RNA provides the 5' m7G cap and first 39 nt for all nuclear mRNAs in kinetoplastids. This small nuclear RNA is transcribed by RNA polymerase II from individual promoters. In Leishmania tarentolae the SL RNA genes reside in two multi-copy tandem arrays designated MINA and MINB. The transcript accumulation from the SL promoter on the drug-selected, episomal SL RNA gene cassette pX-tSL is ~10% that of the genomic array in uncloned L. tarentolae transfectants. This disparity is neither sequence- nor copy-number related, and thus may be due to interference of SL promoter function by epigenetic factors. To explore these possibilities we examined the nucleoplasmic localization of the SL RNA genes as well as their nucleosomal architecture. Results The genomic SL RNA genes and the episome did not co-localize within the nucleus. Each genomic repeat contains one nucleosome regularly positioned within the non-transcribed intergenic region. The 363-bp MINA array was resistant to micrococcal nuclease digestion between the -258 and -72 positions relative to the transcription start point due to nucleosome association, leaving the promoter elements and the entire transcribed region exposed for protein interactions. A pattern of ~164-bp protected segments was observed, corresponding to the amount of DNA typically bound by a nucleosome. By contrast, nucleosomes on the pX-tSL episome were randomly distributed over the episomal SL cassette, reducing transcription factor access to the episomal promoter by approximately 74%. Cloning of the episome transfectants revealed a range of transcriptional activities, implicating a mechanism of epigenetic heredity. Conclusion The disorganized nucleosomes on the pX episome are in a permissive conformation for transcription of the SL RNA cassette approximately 25% of the time within a given parasite. Nucleosome interference is likely the major factor in the apparent transcriptional repression of the SL RNA gene cassette. Coupled with the requirement for run-around transcription that drives expression of the selectable drug marker, transcription of the episomal SL may be reduced even further due to sub-optimal nucleoplasmic localization and initiation complex disruption.

The sequences of minicircle conserved regions from various plant trypanosomatids have been determined and analyzed. The goal of this study was to add another tool to the arsenal of molecular probes for distinguishing between the different trypanosomatids occuring in plants: systemic trypanosomatids multiplying in the sap, those from the laticiferous tubes, and those developing in fruits, seeds or flowers but not in the plant itself and that are frequently considered as opportunistic insect trypanosomatids. As some plant intraphloemic trypanosomatids are the causative agents of important diseases, a clear definition of the different types of trypanosomatids is critical. The conserved region of the mitochondrial minicircle provides several specific features in a small sequence region containing three functionally elements required for minicircle replication. Trees generated from the analysis recapitulated trees drawn from analyses of isoenzymes, RAPD, and particular gene sequences, supporting the validity of the small region used in this work. Three groups of isolates were significant and in accordance with previous work. The peculiarity of phloem-restricted trypanosomatids associated with wilts of coconut and oil palm in Latin America – group H – is confirmed. In agreement with previous studies on their biological and serological properties the results highlighted this group called ‘phloemicola’. It always differentiated from all other latex and fruit isolates or opportunistic trypanosomatids, like insect trypanosomatids. We can assert that phloemicola is the only well-defined taxon among all plant trypanosomatids. A group of non-pathogenic latex isolates from South American euphorbs (G), and a heterogenous group (A) including one fruit, one possible latex and one insect isolate are clearly distinct groups. The group of Mediterranean isolates from latex (D), even with a low boostrap, stood out well from other groups. The remainder of the isolates fell into a heterogeneous cluster. At least eight different groups in the plant trypanosomatids were identified.

In kinetoplastids spliced leader (SL) RNA is trans -spliced onto the 5′ ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m 7 G cap, ribose 2′-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans -spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2′- O -methyltransferases in Trypanosoma brucei . Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A 2 and is not required for subsequent steps; TbMT511 methylates C 3 , without which U 4 methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m 7 G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A 2 , C 3 , and U 4 methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form). Every year 2–3% of these individuals develop severe clinical manifestations (cardiac and digestive forms). In this study a Trypanosoma cruzi DNA microarray was used to compare the transcript profiles of six human isolates: three from asymptomatic and three from cardiac patients. Seven signals were expressed differentially between the two classes of isolates, including tryparedoxin, surface protease GP63, cyclophilin, some hypothetical proteins and the pre-edited maxicircle gene NADH dehydrogenase subunit 7 (ND7). The approximately 30-fold greater signal in cardiac strains for ND7 was the most pronounced of the group, and differential levels of pre-edited ND7 transcript confirmed the microarray analysis. The ND7 gene from asymptomatic isolates showed a deletion of 455 bp from nt 222 to nt 677 relative to ND7 of the CL Brener reference strain. The ND7 gene structure correlated with disease manifestation for 20 isolates from clinically characterised, chronic phase patients. The ND7 lesion produces a truncated product that could impair the function of mitochondrial complex I. Possible links between the integrity of the electron transport chain and symptom presentation are discussed. We propose that ND7 and other genes of the pathway constitute valuable targets for PCR assays in the differential diagnosis of the infective T. cruzi strain. While this hypothesis requires validation by the examination of additional recent parasite isolates from patients with defined pathologies, the identification of specific molecular markers represents a promising advance in the association between parasite genetics and disease pathology.

In kinetoplastids, every nuclear-derived mRNA contains an identical 39-nucleotide (nt) spliced leader at its 5'-terminus. The spliced leader is derived from substrate spliced leader RNA and joined to pre-mRNA by trans-splicing, thus providing mature mRNAs with an m7G cap and additional methylations referred to as cap 4. It was shown previously that mutations spanning nucleotides 10-39 of the spliced leader did not affect substrate spliced leader RNA transcription or trans-splicing in Leishmania tarentolae (Saito, R. M., Elgort, M. G., and Campbell, D. A. (1994) EMBO J. 13, 5460-5469). In this study we examined these sequences for a possible role in translation by assaying the association of mRNAs, which possess mutated spliced leaders, with polysomes. For the nt 28-39 mutated spliced leaders, both the substrate spliced leader RNA and the spliced leader demonstrated a wild-type methylation pattern; spliced nt 28-39 mRNA was found in polysomes. Thus, the nt 28-39 region conserved primary sequence is not a determinant of polysome association. An undermethylated cap 4 structure was present on substrate and mRNA spliced leaders in nt 20-29 mutated exons; nt 20-29 mRNA was not present in polysomes. A differential pattern of cap 4 methylation was seen between the nt 10-19 substrate spliced leader RNA and the nt 10-19 spliced leaders found in the poly(A)+ population of RNA; the nt 10-19 mRNA was not seen in polysomes. Undermethylated spliced leaders did not associate efficiently with polysomes, suggesting a requirement for the cap 4 and/or primary sequence of the spliced leader in translation. This is the first report demonstrating that the spliced leader contains critical structural or sequence determinants for association with polysomes and, hence, translation.

The kinetoplastid protozoan spliced leader (SL) RNA is the common substrate pre-mRNA utilized in all trans -splicing reactions. Here we show by fluorescence in situ hybridization that the SL RNA is present in the cytoplasm of Leishmania tarentolae and Trypanosoma brucei . Treatment with the karyopherin-specific inhibitor leptomycin B was toxic to T. brucei and eliminated the cytoplasmic SL RNA, suggesting that cytoplasmic SL RNA was dependent on the nuclear exporter exportin 1 (XPO1). Ectopic expression of xpo1 with a C506S mutation in T. brucei conferred resistance to leptomycin B. A reduction in SL RNA 3′ extension removal and 5′ methylation of nucleotide U 4 was observed in wild-type T. brucei treated with leptomycin B, suggesting that the cytoplasmic stage is necessary for SL RNA biogenesis. This study demonstrates spatial and mechanistic similarities between the posttranscriptional trafficking of the kinetoplastid protozoan SL RNA and the metazoan cis -spliceosomal small nuclear RNAs.

Addition of a 39-nucleotide (nt) spliced leader (SL) by trans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3′ end of all kinetoplastid SL RNA genes, and that more than six T’s are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3′ ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T’s. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3′ end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3′-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.

Every kinetoplastid mRNA receives a common, conserved leader sequence via the process of trans-splicing. In Leishmania tarentolae the precursor spliced leader RNA is 96 nucleotides, with a 39-nucleotide exon that is 7meG-capped and methylated on the first 4 nucleotides. trans-Splicing was inferred from the presence of tagged leader in the high molecular weight RNA population and confirmed for accuracy by cDNA cloning. Linker scan substitutions within the exon between positions 10 and 39 did not affect trans-splicing. The trans-splicing efficiency for three of the scan exons was proportional to the tagged:wild type ratio in the spliced leader precursor population. Two scan leader RNAs that were efficiently spliced showed reduced methylation. Longer exons showed reduced splicing, whereas 10- or 20-base pair deletions abolished splicing. These results indicate that size, but not content, of the exon is a constraint on the splicing process. These results, in combination with previous data eliminating a role in transcription initiation, suggest that translation may be the selective pressure on the leader content.

The tandem mini-exon gene repeat is an ideal diagnostic target for trypanosomatids because it includes sequences that are conserved absolutely coupled with regions of extreme variability. We have exploited these features and the polymerase chain reaction to differentiate Phytomonas strains isolated from phloem, fruit or latex of various host plants. While the transcribed regions are nearly identical, the intergenic sequences are variable in size and content (130–332 base pairs). The mini-exon genes of these phytomonads can therefore be distinguished from each other and from the corresponding genes in insect trypanosomes, with which they are oft confused.

Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3′ to 5′ editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3′ to 5′ patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3′ to 5′ editing of the COIII mRNA with incorrect guide RNA molecules.

Amplification of DNA sequences from the kinetoplast minicircle DNA was employed as a method for the detection and classification of small numbers of Trypanosoma cruzi cells. Two overlapping fragments from the conserved 120 bp minirepeat regions of the minicircle DNA and one fragment covering the adjacent variable regions were amplified. The minimal amount of minicircle DNA required to detect a product by hybridization with an oligonucleotide probe was 0.015 fg, which represents approximately 10 molecules or 0.1% of the minicircle DNA component of a single cell. The amplification worked equally well with kDNA from several strains of T. cruzi and did not occur with kDNA from several other kinetoplastids. kDNA recovered from less than 10 trypanosomes in whole blood could be used as a template for amplification; the presence of a several billion fold excess of human DNA had no effect on the amplification process. Schizodeme analysis by hybridization with specific oligonucleotides or by direct restriction enzyme digestion could be performed on the amplified fragments representing the minicircle conserved region or variable regions. This method should prove useful as a rapid, specific and sensitive assay for Chagas' disease in chronic patients as well as for epidemiological studies of infected animals and insects.