Alexander A. Kolesnikov

Diversity and phylogeny of insect trypanosomatids based on small subunit rRNA genes: Polyphyly of Leptomonas and Blastocrithidia

Abstract With the aim of further investigating phylogenetic relationships in insect trypanosomatids, we have determined the sequences of small subunit rRNA genes from ten isolates, which were originally classified as Leptomonas, Blastocrithidia, and Wallaceina based on their morphology in the hosts. The inferred maximum likelihood, parsimony, and distance trees indicate that the Leptomonas and Blastocrithidia are polyphyletic, and confirm the polyphyly of Herpetomonas and Crithidia. Blastocrithidia triatoma and Leptomonas collosoma were among the earliest branching lineages among the insect trypanosomatids, while most other isolates were found within a closely related terminal clade, which also included Crithidia fasciculata. This analysis has clearly demonstrated that the morphological classification system of insect trypanosomatids does not always reflect their genetic affinities warranting its revision in the future.

Conservative and divergent base sequence regions in the maxicircle kinetoplast DNA of several trypanosomatid flagellates

The structure of kinetoplast maxicircle DNA from trypanosomatids Crithidia oncopelti, C. luciliae, Leptomonas pessoai and Leishmania gymnodactyli was compared by the blot hybridization method. The sizes of these molecules are 24.5, 34, 31 and 38 kilobase pairs (kbp), respectively. Labelled maxicircle fragments from C. oncopelti were used as probes. A general model of the structural organization of maxicircles is proposed according to which this molecule is composed of a 17kbp conservative region and a divergent one occupying the rest of the molecule. The conservative region contains the sequences homologous to those in all trypanosomatids. The sequence of the divergent region exhibits no cross homology detectable by high stringency hybridization. The main size differences between the maxicircle molecules from different trypanosomatid species are explained by the length variability of their divergent regions.

Diversity of mitochondrial genome organization

In this review, we discuss types of mitochondrial genome structural organization (architecture), which includes the following characteristic features: size and the shape of DNA molecule, number of encoded genes, presence of cryptogenes, and editing of primary transcripts.

Minicircular kinetoplast DNA from Trypanosomatidae

Analysis of primary structure and organization of mitochondrial (kinetoplast) DNA of flagellates occupies a prominent place in the studies of eukaryote mitochondrial genomes, owing to its unusual organization and functioning as well as to the epidemiological role of the Trypanosomatidae family. According to contemporary notions, living zooflagellates are direct descendants of the ancestral forms that gave rise to all eukaryotic kingdoms. Hence, comparative mtDNA studies of recent Trypanosomatidae open broad prospects for phylogenetic reconstructions and analysis of presumable routes of eukaryote evolution. The structure, characteristics, and functions of Trypanosomatidae minicircular kinetoplast DNA are discussed here.

Comparative Restriction Enzyme Cleavage Analysis of Kinetoplast DNA from the Lower Trypanosomatids Isolated in the North-West Region of the USSR

Summary The usefulness of molecular parameters of kinetoplast DNA (kDNA) as taxonomic criteria in the systematics of lower trypanosomatids was studied. 18 previously isolated lower trypanosomatid strains were classified on the basis of their minicircle DNA size and the maxicircle and minicircle cleavage profiles generated with restriction endonucleases. The results of such a classification were compared with the taxonomic status of isolates previously determined with the aid of the traditional systematics criteria. On the basis of cleavage data the isolates were grouped into four distinctly different schizodemes. The first one (A) comprised two Blastocrithidia species, whereas in the second schizodeme (B) three Leptomonas species were contained, thus revealing a good correspondence at the generic level between the 2 types of criteria. The schizodeme D was composed of a single Leptomonas species, which previously was found to differ significantly from the above mentioned three Leptomonas species by the traditional criteria. The most extensive schizodeme C included 12 isolates with a similar though not identical kDNA structure. Previously these isolates were referred to three different genera — Leptomonas, Crithidia and Blastocrithidia . The taxonomic status of several isolates has been determined presumptively. The extent of variability in kDNA structure within this schizodeme was not profound, being scarcely consistent with the different generic status of the isolates. To explain this discrepancy, the different possibilities including unspecific invasions were suggested. The results presented in this study clearly show that the restriction endonuclease analysis of kDNA may be employed as a useful and sensitive tool in developing the lower trypanosomatid taxonomy.

Conserved repeats in the kinetoplast maxicircle divergent region of Leishmania sp. and Leptomonas seymouri

The maxicircle control region [also termed divergent region (DR)] composed of various repeat elements remains the most poorly studied part of the kinetoplast genome. Only three extensive DR sequences demonstrating no significant similarity were available for trypanosomatids (Leishmania tarentolae, Crithidia oncopelti, Trypanosoma brucei). Recently, extensive DR sequences have been obtained for Leishmania major and Trypanosoma cruzi. In this work we have sequenced DR fragments of Leishmania turanica, Leishmania mexicana, Leishmania chagasi and two monogenetic trypanosomatids Leptomonas seymouri and Leptomonas collosoma. With the emergence of the additional extensive sequences some conserved features of DR structure become evident. A conserved palindromic sequence has been revealed in the DRs of the studied Leishmania species, L. seymouri, and T. cruzi. The overall DR structure appears to be similar in all the Leishmania species, their relative L. seymouri, and T. brucei: long relatively GC-rich repeats are interspersed with clusters of short AT-rich repeats. C. oncopelti, L. collosoma, and T. cruzi have a completely different DR structure. Identification of conserved sequences and invariable structural features of the DR may further our understanding of the functioning of this important genome fragment.

Reduction of the Edited Domain of the Mitochondrial A6 Gene for ATPase Subunit 6 in Trypanosomatidae

The sequence of mitochondrial A6 (MURF4) was compared for several trypanosomatids in order to assess the reduction of the edited domain (ED). The association between the ED reduction and the phylogenetic position of a species proved to be less tight than believed earlier. Compared with digenetic species, monogenetic ones more often displayed ED reduction and had smaller ED.

The mitochondrial ND8 gene from Crithidia oncopelti is not pan-edited

RNA editing in trypanosomatid mitochondria is a process involving the insertion and deletion of uridine residues within the coding region of maxicircle messenger RNA transcripts. Twelve of the 17 known genes need editing to produce functional molecules. We have analyzed the predicted editing sites for the Crithidia oncopelti mitochondrial NADH‐ubiquinone oxidoreductase subunit 8 (ND8) gene based on known mRNAs from other trypanosomatid species. All studied ND8 mRNAs undergo editing throughout the coding (and 3′ non‐coding) sequences (pan‐editing). The 5′ part of the C. oncopelti ND8 gene undergoes editing (like in Leishmania tarentolae and Trypanosoma brucei) while the 3′ part of the pre‐edited gene corresponds to the 3′ part of edited ND8 mRNAs from other organisms. The organization of the ND8 gene in C. oncopelti mitochondrial DNA differs from all organisms investigated so far – this gene is not pan‐edited. We have also localized the guide RNA for cytochrome b between 9S rRNA and the ND8 gene. This RNA shows high homology to the gCYb‐II gene of L. tarentolae and the gCyb gene of Crithidia fasciculata. A hypothetical editing pattern for the cytochrome b gene in C. oncopelti maxicircles is proposed.

Trypanosomatid mitochondrial RNA editing: Dramatically complex transcript repertoires revealed with a dedicated mapping tool

Abstract RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3′ to 5′ on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. We also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.

The mitochondrial genome. The nucleoid

Mitochondrial DNA (mtDNA) in cells is organized in nucleoids containing DNA and various proteins. This review discusses questions of organization and structural dynamics of nucleoids as well as their protein components. The structures of mt-nucleoid from different organisms are compared. The currently accepted model of nucleoid organization is described and questions needing answers for better understanding of the fine mechanisms of the mitochondrial genetic apparatus functioning are discussed.