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Symbionts are adapted to work with corals Many corals have formed mutualistic associations with dinoflagellate symbionts, which are thought to provide nutrients and other benefits. To examine the underlying genetics of this association, S. Lin et al. sequenced the genome of the endosymbiont dinoflagellate Symbiodinium kawagutii. The genome includes gene number expansions and encodes microRNAs that show complementarity to genes within the coral genome. Such microRNAs may be involved in regulating coral genes. Furthermore, coral and S. kawagutii appear to share homologs of genes encoding specific nutrient transporters. The findings shed light on how symbiosis is established and maintained between dinoflagellates and corals. Science , this issue p. 691

Trypanosomatids are parasitic protozoans characterized by several unique structural and metabolic processes that include exquisite mechanisms associated with gene expression and regulation. During the initiation of protein synthesis, for instance, mRNA selection for translation seems to be mediated by different eIF4F-like complexes, which may play a significant role in parasite adaptation to different hosts. In eukaryotes, the heterotrimeric eIF4F complex (formed by eIF4E, eIF4G, and eIF4A) mediates mRNA recognition and ribosome binding and participates in various translation regulatory events. Six eIF4Es and five eIF4Gs have been described in trypanosomatids with several of these forming different eIF4F-like complexes. This has raised questions about their role in differential mRNA translation. Here we have studied further TbEIF4E2, the least known eIF4E homologue from Trypanosoma brucei, and found that it is not associated with an eIF4G homolog. It is, however, associated with mature mRNAs and binds to a histone mRNA stem-loop-binding protein (SLBP), one of two Trypanosoma SLBP homologs (TbSLBP1 and TbSLBP2). TbSLBP1 is more similar to the mammalian counterpart while TbSLBP2 is exclusive to trypanosomatids and related organisms. TbSLBP2 binds to TbEIF4E2 through a conserved central region missing in other SLBP homologs. Both SLBPs, as well as TbEIF4E2, were found to localize to the cytoplasm. TbEIF4E2 and TbSLBP2 are differentially expressed during cell culture, being more abundant in early-log phase, with TbSLBP2 also showing cell-cycle dependent expression. The new data reinforce unique aspects of the trypanosomatid eIF4Es, with the TbEIF4E2–TbSLBP complex possibly having a role in differential selection of mRNAs containing stem-loop structures.

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.

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form, IF). Each year, approximately 3% of them develop lesions in the heart or gastrointestinal tract. Cardiomyopathy (CCHD) is the most severe manifestation of Chagas disease. The factors that determine the outcome of the infection are unknown, but certainly depend on complex interactions amongst the genetic make-up of the parasite, the host immunogenetic background and environment. In a previous study we verified that the maxicircle gene NADH dehydrogenase (mitochondrial complex I) subunit 7 (ND7) from IF isolates had a 455 bp deletion compared with the wild type (WT) ND7 gene from CCHD strains. We proposed that ND7 could constitute a valuable target for PCR assays in the differential diagnosis of the infective strain. In the present study we evaluated this hypothesis by examination of ND7 structure in parasites from 75 patients with defined pathologies, from Southeast Brazil. We also analysed the structure of additional mitochondrial genes (ND4/CR4, COIII and COII) since the maxicircle is used for clustering Trypanosoma cruzi strains into three clades/haplogroups. We conclude that maxicircle genes do not discriminate parasite populations which induce IF or CCHD forms. Interestingly, the great majority of the analysed isolates belong to T. cruzi II (discrete typing unit, (DTU) IIb) genotype. This scenario is at variance with the prevalence of hybrid (DTU IId) human isolates in Bolivia, Chile and Argentina. The distribution of WT and deleted ND7 and ND4 genes in T. cruzi strains suggests that mutations in the two genes occurred in different ancestrals in the T. cruzi II cluster, allowing the identification of at least three mitochondrial sub-lineages within this group. The observation that T. cruzi strains accumulate mutations in several genes coding for complex I subunits favours the hypothesis that complex I may have a limited activity in this parasite.

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.

Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

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.

Genetic variation in Trypanosoma cruzi is likely a key determinant in transmission and pathogenesis of Chagas disease. We have examined nine loci as markers for the extant T. cruzi strains. Four distinct alleles were found for each locus, corresponding to the sequence classes present in the homozygous discrete typing units (DTUs) I, IIa, IIb, and IIc. The alleles in DTUs IIa and IIc showed a spectrum of polymorphism ranging from DTU I-like to DTU IIb-like, in addition to DTU-specific sequence variation. DTUs IId and IIe were indistinguishable, showing DTU homozygosity at one locus and heterozygosity with DTU IIb and IIc allelic sequences at eight loci. Recombination between the DTU IIb and IIc alleles is evidenced from mosaic polymorphisms. These data imply that two discrete hybridization events resulted in the formation of the current DTUs. We propose a model in which a fusion between ancestral DTU I and IIb strains gave rise to a heterozygous hybrid that homogenized its genome to become the homozygous progenitor of DTUs IIa and IIc. The second hybridization between DTU IIb and IIc strains that generated DTUs IId and IIe resulted in extensive heterozygosity with subsequent recombination of parental genotypes.

The spliced leader RNA gene (SL RNA) repeat is present in large multicopy arrays and has been used as a marker for the diversity of kinetoplastid protozoans. Intra-array variation could affect conclusions made using a randomly isolated repeat as a marker. We examined the Leishmania major (Friedlin) and Trypanosoma cruzi (CL Brener) genome projects for SL RNA repeat sequences in order to assess their homogeneity and the possible effects of sequence variation on taxonomic interpretation. Of the dozens of distinct sequence classes examined, no single copy would bias clustering analyses with regard to other closely related species or isolates. Six dimorphic sites within the T. cruzi transcribed region were found to be linked and are predicted to yield a heterogeneous SL RNA population. The variation that exists among the repeats paints a picture of the broad mechanisms of array maintenance and evolution where site-specific mutations in a single repeat may be spread throughout the array and recombined with existing repeats to create new sequence classes, all occurring under selective pressure to maintain or increase the fitness of the cell line in which these events occur.

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.

The biodiversity of insect trypanosomes is largely unknown, resulting in significant gaps in the understanding of pathogen evolution. A culture-independent preliminary survey of trypanosomatid fauna was conducted for the parasites of Heteroptera (Hemiptera) from several localities in Costa Rica. Trypanosomatid infections were detected by light microscopy of smeared gut contents. Out of 257 insects representing 6 families, infections were found in 62 cases; cultures were obtained for 29 new isolates. Gut material from infected hosts was preserved in the field using an SDS–EDTA buffer solution for subsequent DNA extraction in the laboratory. PCR amplification of the trypanosomatid-specific spliced leader (SL) RNA gene repeats was successful for 60 field samples. Eighteen distinct SL RNA typing units were identified in a set of 28 samples analysed in detail. Cluster analysis indicated that these typing units were unique and thus could represent new species and, in some cases, new genera. This study reveals only a minor fraction of the trypanosomatid biodiversity, which is anticipated to be high.

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.

We have determined the sequences of 5S rRNA and spliced leader (SL) RNA genes, and adjacent intergenic regions for representatives of all known trypanosomatid genera parasitizing insects. The genetic loci have been analyzed separately as well as by a combined approach. Several isolates, assigned by morphology to different genera (Leptomonas spp., Blastocrithidia spp.), seem to belong to a single species with an unexpectedly wide host and geographical range. An unnamed trypanosomatid isolated from rats in Egypt was found to belong to the genus Herpetomonas, so far associated with insect hosts only. It is closely related to Herpetomonas ztiplika, a parasite of a blood-sucking biting midge. Apparently several different trypanosomatid species can infect one insect species, as exemplified by Leptomonas sp. PL and Wallaceina sp. Wsd, which were isolated from different specimens of Salda littoralis on the same locality and day. However, since the same species of Leptomonas was obtained from insect hosts belonging to different genera, some insect trypanosomatids may have low host specificity. Our data revealed additional discrepancies between molecular phylogenetic data and cell morphology, rendering current trypanosomatid taxonomy unreliable.

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.

Transcription in the kinetoplastid protozoa shows substantial variation from the paradigms of eukaryotic gene expression, including polycistronic transcription, a paucity of RNA polymerase (RNAP) II promoters, no qualitative regulated transcription initiation for most protein-coding genes, transcription of some protein-coding genes by RNAP I, an exclusive subnuclear location for VSG transcription, the dependence of small nuclear RNA gene transcription on an upstream tRNA gene, and the synthesis of mitochondrial tRNAs in the nucleus. Here, we present a broad overview of what is known about transcription in the kinetoplastids and what has yet to be determined.

Trypanosomes isolated from South American bats include the human pathogen Trypanosoma cruzi. Other Trypanosoma spp. that have been found exclusively in bats are not well characterized at the DNA sequence level and we have therefore used the SL RNA gene to differentiate and characterize kinetoplastids isolated from bats in South America. A Trypanosoma sp. isolated from bats in southern Brazil was compared with the geographically diverse isolates T. cruzi marinkellei, T. vespertilionis, and T. dionisii. Analysis of the SL RNA gene repeats revealed size and sequence variability among these bat trypanosomes. We have developed hybridization probes to separate these bat isolates and have analysed the DNA sequence data to estimate their relatedness. A new species, Trypanosoma desterrensis sp. n., is proposed, for which a 5S rRNA gene was also found within the SL RNA repeat.

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.

There is a high level of genetic diversity among the clinical isolates of the taxon Trypanosoma cruzi. The results of recent studies on well defined gene loci have revealed that T. cruzi can be divided into two major phylogenetic lineages, designated T. cruzi I and T. cruzi II. Further intra-lineage polymorphisms were explored, in clinical isolates from five representative regions of Brazil, using the internal transcribed spacers that flank the 5.8S ribosomal RNA gene. These regions were amplified in a PCR and the products were digested with six restriction enzymes before being subjected to RFLP analysis in polyacrylamide gels. The results were used to construct a complex evolutionary tree, in which the isolates were clustered into two major phylogenetic lineages with a low level of similarity. To investigate the evolutionary relationships between the isolates, the PCR products were cloned and sequenced. The results of the subsequent phylogenetic analysis confirmed the presence of the two major groups of T. cruzi and showed that zymodeme 3, a third iso-enzymatic sub-group, is closer to T. cruzi I than to T. cruzi II.

To establish the relationships of the lizard- and mammal-infecting Leishmania, we characterized the intergenic spacer region of ribosomal RNA genes from L. tarentolae and L. hoogstraali. The organization of these regions is similar to those of other eukaryotes. The intergenic spacer region was approximately 4 kb in L. tarentolae and 5.5 kb in L. hoogstraali. The size difference was due to a greater number of 63-bp repetitive elements in the latter species. This region also contained another element, repeated twice, that had an inverted octanucleotide with the potential to form a stem-loop structure that could be involved in transcription termination or processing events. The ribosomal RNA gene localization showed a distinct pattern with one chromosomal band (2.2 Mb) for L. tarentolae and two (1.5 and 1.3 Mb) for L. hoogstraali. The study also showed sequence differences in the external transcribed region that could be used to distinguish lizard Leishmania from the mammalian Leishmania. The intergenic spacer region structure features found among Leishmania species indicated that lizard and mammalian Leishmania are closely related and support the inclusion of lizard-infecting species into the subgenus Sauroleishmania proposed by Saf'janova in 1982.

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.

In recent years, much has been learned about the cis-elements controlling transcription of the kinetoplastid spliced leader (SL) RNA gene. The SL RNA gene contains the first 39 nucleotides that are trans-spliced on to all nuclear-derived mRNAs in these organisms. Transcription initiation is determined by two precisely spaced upstream elements and transcription termination is directed by the downstream poly-T tract, although the RNA polymerase responsible for SL RNA synthesis is still questioned. In this article, David Campbell, Nancy Sturm and Michael Yu review the field of kinetoplastid SL RNA gene transcription, address past proposals in light of current data and discuss some of the differences that appear in the literature.

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.

The spliced leader (SL) RNA is a defining element in the gene expression of kinetoplastids. The first 39 nt of this small RNA are trans-spliced onto every nuclear message, providing a unique hypermethylated cap and sequence elements required for stability and translation. Transcribed from a large tandem array, the journey that each primary SL transcript takes en route to splicing is marked by molecular modifications. Methylation, pseudouridylation, and 3′-end nuclease processing contribute to the mature product. The consequences of this elaborate pathway are not understood fully, but may reveal distinctions that will make these oft-parasitic organisms yield their foothold in the vertebrate host.

0. Fernandesi*3, S. S. Santos’, E. CupoUlo’, B. Mendonga’, R. Derre3, A. C. V. Junqueira’, L. C. Santos’, N. R. Sturm4, R. l3. NaifP, T. V. Barret’, Il. A. Campbell4 and J. R. Coura’ ‘Department of Tropical Medicine and 2Department of Imw~nunology, Instiwo OswaEdo Cruz, Fiocruz, Rio de Janeiro, Brazil; 3DepaPtment of Pathology, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil; 4Department of Microbiology, InwunoZogy and MolecuZar Genetics, Uv 51nstituto National de Pesquisas da AmazBnia, INPA, Manaus, Brazil Keywords:Americantrypanosomiasis, Chagasdisease

RNA editing in the mitochondria of kinetoplastid protoza involves the insertion and/or deletion of precise numbers of uridine residues at precise locations in the numbers of uridine residues at precise locations in the transcribed RNA of certain genes. These genes are known as cryptogenes. In this paper we study computational algorithms to search for unknown cryptogenes and for the associated templates for insertion of uridines, gRNA sequences. The pairwise similarity search algorithm of Smith and Waterman (1) is modified to study this problem. The algorithm searches for unknown gRNAs given the cryptogene sequence. The method is tested on 4 known cryptogenes from L.tarentolae which are known to have 7 associated gRNAs. The statistical distribution of the longest gRNA when comparing random sequences is derived. Finally we develop an algorithm to search for cryptogenes using amino acid sequences from related proteins.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report. Universidade de São Paulo Instituto de Química Departamento de Bioquímica Fundação Oswaldo Cruz-Fiocruz Centro de Pesquisas Gonçalo Moniz Universidade Federal de São Paulo (UNIFESP) Imunologia e Parasitologia Departamento de Microbiologia University of California David Geffen School of Medicine Department of Microbiology Instituto Oswaldo Cruz-Fiocruz Laboratório Interdisciplinar de Pesquisas Médicas Universidad de los Andes Departamento de Ciencias Biológicas Facultad de Ciencias Universidade Federal do Triângulo Mineiro Departamento de Ciências Biológicas Universidade Federal de Minas Gerais Instituto de Ciências Biológicas Departamento de Bioquímica e Imunologia London School of Hygiene and Tropical Medicine Pathogen Molecular Biology Unit Instituto de Pesquisas René Rachou-Fiocruz UMR Laboratory of Genetics and Evolution of Infectious Diseases Instituto de Ingeniería Genética y Biología Molecular Laboratorio de Biología Molecular de la Enfermedad de Chagas UNIFESP, Imunologia e Parasitologia Depto. de Microbiologia SciELO

Infections by Trypanosoma cruzi are transmitted commonly by triatomines, hematophagous insects adapted to anthropophilic behavior. With its potential enzootic presence for over 90 million years, Chagas disease in humans has been documented in 9 thousand-year-old mummies from the Atacama Desert. Lately, Chagas disease has shown exponential growth because European and African colonizers dwelling in huts infested by triatomines contaminated with T. cruzi were promptly infected. Nowadays, Chagas disease affects 18 million people and is considered the most lethal endemic infectious disease in the Western Hemisphere.

As a parasite with a potentially decades-long intracellular stage, Trypanosoma cruzi could represent a case study for extreme mechanisms for the maintenance of mitochondrial sequence integrity. While an intact mitochondrial repertoire of membrane-associated cytochromes and NADH dehydrogenases are required for passage through the insect forms, within the vertebrate host many of these products may be dispensable, as in vertebrate-exclusive relatives. The degeneration seen in maxicircle genomes from clinical isolates and from culture indicates that the maintenance of intact maxicircles and a complete library of minicircles is an uphill battle that the parasite manages actively. Both the maxicircle and minicircle have been implicated in the clinical manifestation of Chagas disease. The implications of both these scenarios on the biology of the parasite relative to the host are discussed.

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.

The genetic palette from which the spectrum of variability in Trypanosoma cruzi has been drawn is astonishingly limited. In this review we address the roots of this unusual pedigree and the usefulness of various taxonomic markers in relation to the manifestation of clinical disease and the geographic distribution of the parasite. The circumstances leading to the population structure of the extant strains were dictated by the unusual and apparently exceedingly rare mode of genetic exchange employed in this species, that being the non-meiotic fusion of two diploid cells. Two-hybridization events have been postulated in the whole of the T. cruzi pedigree, the first of which yielded the four predominant nuclear genotypes. Hybridization may be a common occurrence among closely related strains of T. cruzi, but either infrequent or inefficient when two diverse strains attempt the process. Two of the genotypes define the parental lineages, while the other two are mosaics of the parental contributions distinguished from one another by polymorphisms accumulated after the separation of a common, homozygous hybrid progeny line. The greatest genetic complexity is seen in the result of the second fusion event between one of the original parental strains and a progeny strain. The second generation of progeny reveals the proximal consequences of fusion, maintaining widespread nuclear heterozygosity and the first examples of recombination between the genotypes involved in the second hybridization. If the genesis of the heterozygous progeny follows the same path as their predecessors, these lines will move toward homozygosity after having had the opportunity for recombination. Thus, the total number of alleles may increase to five in another few million years.

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.

Chimeric RNA molecules were detected by polymerase chain reaction amplification of kinetoplast RNA using a 3' primer specific to mRNA and a 5' primer specific to guide RNA (gRNA), and directly by Northern analysis. Covalent linkage of the 3' oligo(U) tail of the gRNA to the mRNA occurs at editing sites. Chimeric molecules were isolated for NADH dehydrogenase subunit 7 and cytochrome oxidase subunits II and III. We propose that these molecules are intermediates in the editing process and that successive transesterifications result in the transfer of uridine residues from the gRNA 3' oligo(U) tail to an editing site, with the number of uridine residues determined by base pairing with adenine and guanine "guide" nucleotides in the gRNA.