Larry Simpson

Phylogeny of trypanosomes as inferred from the small and large subunit rRNAs: implications for the evolution of parasitism in the trypanosomatid protozoa☆

Sequences of the small rRNA genes and partial sequences of the large rRNA genes were obtained by PCR amplification from a variety of vertebrate trypanosomes. The trypanosome species and hosts included Trypanosoma avium from a bird, T. rotatorium from an amphibian, T. boissoni from an elasmobranch, T. triglae from a marine teleost and T. carassii from a freshwater teleost. Phylogenetic relationships among these species and other representatives of the family Trypanosomatidae were inferred using maximum likelihood, maximum parsimony and evolutionary parsimony. The trypanosomatid tree was rooted using rRNA sequences from two species from the suborder Bodonina. All methods showed that the mammalian parasite, Trypanosoma brucei, constitutes the earliest divergent branch. The remaining trypanosomes formed a monophyletic group. Within this group, the bird trypanosome was grouped with T. cruzi, while the elasmobranch trypanosome and the two fish trypanosome species formed a group with an affinity to T. rotatorium. Our results provide no evidence for co-evolution of trypanosomatids and their hosts, either vertebrate or invertebrate. This suggests that evolution of trypanosomatids was accompanied by secondary acquisitions of hosts and habitats.

The Kinetoplast of the Hernoflagellates

Publisher Summary Several groups of flagellated protozoa contain a unique mitochondrion, which has an exceptionally large amount of DNA localized in one region of the inner matrix. The term “kinetoplast” signifies the portion of the mitochondrion containing the mitochondrial DNA, including the enclosing mitochondrial membranes. Kinetoplast DNA (K-DNA) indicates the DNA localized in the inner matrix of the mitochondrion that stains with basic dyes. The existence of one mitochondrion per cell should be conclusively proved by the three-dimensional reconstruction technique. This helps in interpreting the nature of the mitochondrial changes in the slender and stumpy bloodstream trypomastigotes in dye-induced dyskinetoplastic forms. The replication of K-DNA should be investigated, both in terms of the intracellular regulatory mechanisms involved in the synchrony of the kinetoplast S period with the nuclear S period and the actual replication of the minicircles and long DNA molecules.

RNA editing and the mitochondrial cryptogenes of kinetoplastid protozoa

Le RNA editing est une modification post-transcriptionnelle des mRNA. Il consiste en une addition de residus de base unidine a differentes positions du messager. Il est observe sur le RNA mitochondrial des trypanosomes au niveau de leur kinetoplaste. On peut le constater aussi sur 3 autres especes: L. tarentolae, T. brucei et C. fasciculata. 2 enzymes seraient les responsables: par modification de la structure primaire codante, elles sont obligatoirement impliquees dans la regulation du metabolisme

Isolation of a U‐insertion/deletion editing complex from Leishmania tarentolae mitochondria

A multiprotein, high molecular weight complex active in both U‐insertion and U‐deletion as judged by a pre‐cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem affinity purification (TAP) procedure, using three different TAP‐tagged proteins of the complex. This editing‐ or E‐complex consists of at least three protein‐containing components interacting via RNA: the RNA ligase‐containing L‐complex, a 3′ TUTase (terminal uridylyltransferase) and two RNA‐binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identified by mass spectrometric analysis of the core L‐complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids.

C to U editing of the anticodon of imported mitochondrial tRNATrp allows decoding of the UGA stop codon in Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNATrp(CCA) can decode the standard UGG tryptophan codon but can not decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNATrp undergoes a specific C to U nucleotide modification in the first position of the anticodon, which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non‐universal tRNA by compartmentalizing an editing activity that modifies the anticodon of the imported tRNA.

Kinetoplast DNA in Trypanosomid Flagellates

Publisher Summary This chapter discusses the kinetoplast DNA in trypanosomid flagellates. The chapter reviews the recent developments in the application of recombinant DNA technology to problems of the structure, replication, and transcription of the unusual mitochondrial DNA in the kinetoplastid protozoa known as “kinetoplast DNA.” Kinetoplast DNA is discussed in terms of its two molecular components—minicircles and maxicircles, with an emphasis on species-dependent variations. The chapter focuses on the general characteristics of minicircle DNA of several species, including Trypanosoma brucei, Trypanosoma equiperdum, Leishmania tarentolae, Crithidia fasciculata, and Crithidia luciliae. Unlike most other kinetoplastid species, the kDNA minicircles from T.equiperdum are homogeneous in base sequence. The minicircles in the Leishmania are the smallest reported to date. L .tarentolae minicircles are about 870 bp in size and show extensive sequence heterogeneity by restriction enzyme analysis. The minicircles in the Leishmania are the smallest reported to date. L.tarentolae minicircles are about 870 bp in size and show extensive sequence heterogeneity by restriction enzyme analysis. The maxicircle component of the kinetoplast DNA appears to represent the homolog of the informational mitochondria1 DNA found in other eukaryotic cells.

A tale of two TUTases

The insertion and deletion of U residues at specific sites in mRNAs in trypanosome mitochondria is thought to involve 3′ terminal uridylyl transferase (TUTase) activity. TUTase activity is also required to create the nonencoded 3′ oligo[U] tails of the transacting guide RNAs (gRNAs). We have described two TUTases, RET1 (RNA editing TUTase 1) and RET2 (RNA editing TUTase 2) as components of different editing complexes. Tandem affinity purification-tagged Trypanosoma brucei RET2 (TbRET2) was expressed and localized to the cytosol in Leishmania tarentolae cells by removing the mitochondrial signal sequence. Double-affinity isolation yielded tagged TbRET2, together with a few additional proteins. This material exhibits a U-specific transferase activity in which a single U is added to the 3′ end of a single-stranded RNA, thereby confirming that RET2 is a 3′ TUTase. We also found that RNA interference of RET2 expression in T. brucei inhibits in vitro U-insertion editing and has no effect on the length of the 3′ oligo[U] tails of the gRNAs, whereas down-regulation of RET1 has a minor effect on in vitro U-insertion editing, but produces a decrease in the average length of the oligo[U] tails. This finding suggests that RET2 is responsible for U-insertions at editing sites and RET1 is involved in gRNA 3′ end maturation, which is essential for creating functional gRNAs. From these results we have functionally relabeled the previously described TUT-II complex containing RET1 as the guide RNA processing complex.

Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNAGlu(UUC) and tRNAGln(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity‐isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5‐methoxycarbonylmethyl‐2‐ thiouridine (mcm5s2U), whereas mt tRNAs have 5‐ methoxycarbonylmethyl‐2′‐O‐methyluridine (mcm5Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5‐methoxycarbonylmethyluridine (mcm5U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria‐specific tRNALys(UUU) from the cytosol and found mcm5U at its wobble position, while its mitochondrial counterpart has mcm5Um. Mt tRNALys and in vitro transcribed tRNAGlu were imported much more efficiently into isolated mitochondria than the native cy tRNAGlu in an in vitro importation experiment, indicating that cytosol‐specific 2‐thiolation could play an inhibitory role in tRNA import into mitochondria.

Isolation and characterization of kinetoplast DNA from Leishmania tarentolae

Abstract Kinetoplast DNA (ϱ = 1.703 g/ml.) was isolated by preparative cesium chloride ultracentrifugation in a fixed-angle rotor from total cell DNA of Leishmania tarentolae and examined in terms of sedimentation properties, melting characteristics, and appearance in the electron microscope. It consisted of several molecular types, either free or bound together in associations of variable size: minicircles (molecular weight = 0.56 ± 0.03 × 106), catenated minicircles, “figure 8” molecules, and long molecules. The associations seem to be held together by the long molecules threading through the smaller circles and catenanes. The large associations could be broken down by sonication, DNase II-treatment, or shear forces. Minicircles, catenated dimers, trimers, and small linear fragments were separated on preparative sucrose gradients of sonicated DNA, and S20,w values were assigned to each molecular type by band sedimentation in the analytical ultracentrifuge. The kinetoplast DNA was found to be conserved during at least 4.8 cell divisions. There are more than 14 DNA units which segregate independently of each other on cell division. Nuclear DNA (ϱ = 1.716 g/ml.) consisted of long, linear molecules, and had a monophasic melting curve. Kinetoplast DNA did not denature irreversibly until the closed circular molecules were nicked or broken. A preliminary model was presented for the molecular architecture of the kinetoplast DNA structure in situ from the fact that the width of the kinetoplast DNA found in thin sections is equal to the diameter of a minicircle.

Evolution of parasitism: kinetoplastid protozoan history reconstructed from mitochondrial rRNA gene sequences.

A phylogenetic tree for the evolution of five representative species from four genera of kinetoplastid protozoa was constructed from comparison of the mitochondrial 9S and 12S rRNA gene sequences and application of both parsimony and evolutionary parsimony algorithms. In the rooted version of the tree, the monogenetic species Crithidia fasciculata is the most deeply rooted, followed by another monogenetic species, Leptomonas sp. The three digenetic species Trypanosoma cruzi, Trypanosoma brucei, and Leishmania tarentolae branch from the Leptomonas line. The substitution rates for the T. brucei and T. cruzi sequences were 3-4 times greater than that of the L. tarentolae sequences. This phylogenetic tree is consistent with our cladistic analysis of the biological evidence including life cycles for these five species. A tentative time scale can be assigned to the nodes of this tree by assuming that the common ancestor of the digenetic parasites predated the separation of South America and Africa and postdated the first fossil appearance of its host (inferred by parsimony analysis). This time scale predicts that the deepest node occurred at 264 +/- 51 million years ago, at a time commensurate with the fossil origins of the Hemiptera insect host. This implies that the ancestral kinetoplastid and its insect host appeared at approximately the same time. The molecular data suggest that these eukaryotic parasites have an evolutionary history that extends back to the origin of their insect host.