Kinetoplast Maxicircle Research Articles

Identification of a unique conserved region from a kinetoplastid genome of Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2 in Thailand

Mitochondrial DNAs (mtDNAs) appear in almost all eukaryotic species and are useful molecular markers for phylogenetic studies and species identification. Kinetoplast DNAs (kDNAs) are structurally complex circular mtDNA networks in kinetoplastids, divided into maxicircles and minicircles. Despite several kDNAs of many Leishmania species being examined, the kDNAs of the new species, Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2, have not been explored. This study aimed to investigate the maxicircle and minicircle DNAs of L. orientalis strain PCM2 using hybrid genome sequencing technologies and bioinformatic analyses. The kDNA sequences were isolated and assembled using the SPAdes hybrid assembler from the Illumina short-read and PacBio long-read data. Circular contigs of the maxicircle and minicircle DNAs were reconstructed and confirmed by BLASTn and rKOMICs programs. The kDNA genome was annotated by BLASTn before the genome comparison and phylogenetic analysis by progressiveMauve, MAFFT, and MEGA programs. The maxicircle of L. orientalis strain PCM2 (18,215 bp) showed 99.92% similarity and gene arrangement to Leishmania enriettii strain LEM3045 maxicircle with variation in the 12s rRNA gene and divergent region. Phylogenetics of the whole sequence, coding regions, divergent regions, and 12s rRNA gene also confirmed this relationship and subgenera separation. The identified 105 classes of minicircles (402–1177 bp) were clustered monophyletically and related to the Leishmania donovani minicircles. The kinetoplast maxicircle and minicircle DNAs of L. orientalis strain PCM2 contained a unique conserved region potentially useful for specific diagnosis of L. orientalis and further exploration of this parasite population genetics in Thailand and related regions.

Novel insertions in the mitochondrial maxicircle of Trypanosoma musculi, a mouse trypanosome.

Trypanosoma musculi is a, globally distributed, mouse-specific haemoflagellate, of the family Trypanosomatidae, which shares similar characteristics in morphology with Trypanosoma lewisi. The kinetoplast (mitochondrial) DNA of Trypanosomatidae flagellates is comprised of catenated maxicircles and minicircles. However, genetic information on the T. musculi kinetoplast remains largely unknown. In this study, the T. musculi maxicircle genome was completely assembled, with PacBio and Illumina sequencing, and the size was confirmed at 34 606 bp. It consisted of 2 distinct parts: the coding region and the divergent regions (DRs, DRI and II). In comparison with other trypanosome maxicircles (Trypanosoma brucei, Trypanosoma cruzi and T. lewisi), the T. musculi maxicircle has a syntenic distribution of genes and shares 73.9, 78.0 and 92.7% sequence identity, respectively, over the whole coding region. Moreover, novel insertions in MURF2 (630 bp) and in ND5 (1278 bp) were found, respectively, which are homologous to minicircles. These findings support an evolutionary scenario similar to the one proposed for insertions in Trypanosoma cruzi, the pathogen of American trypanosomiasis. These novel insertions, together with a deletion (281 bp) in ND4, question the role of Complex I in T. musculi. A detailed analysis of DRII indicated that it contains numerous repeat motifs and palindromes, the latter of which are highly conservative and contain A5C elements. The comprehensively annotated kinetoplast maxicircle of T. musculi reveals a high degree of similarity between this parasite and the maxicircle of T. lewisi and suggests that the DRII could be a valuable marker for distinguishing these evolutionarily related species.

Phylogenetic insights on Leishmania detected in cats as revealed by nucleotide sequence analysis of multiple genetic markers

Cats have been found infected by the same Leishmania species that also infect dogs and humans in both the New and Old Worlds, and their role as additional reservoir hosts of L. infantum has been previously suggested. Currently, the genetic diversity of Leishmania spp. detected in cats is poorly understood. In this cross-sectional study, the partial nucleotide sequences of four gene markers (cytB, g6pdh, hsp70 and ITS-rDNA) were explored to investigate the genetic diversity and the phylogenetic relationships of Leishmania parasites detected in cats. A total of 25 cat buffy coat samples where the presence of Leishmania SSU-rDNA was revealed by PCR (from a convenience sample of 465 cats screened), as well as six Leishmania strains previously isolated from cats, were included in this study. Phylogenetic analyses showed that the majority of Leishmania parasites detected in cats did not display distinctive genetic features, sharing the same genetic types with L. infantum strains isolated from humans, dogs and phlebotomine sand flies. Unexpectedly, DNA of L. major and/or of a L. major/L. donovani sensu lato hybrid was detected in buffy coat samples of two cats from different regions of Portugal. However, a mix infection hypothesis cannot be formally excluded. To our knowledge, this study represents the first evidence for the presence of DNA of Leishmania hybrid parasites in cats. The results reported here not only reinforce the idea that cats play a role in the epidemiology of zoonotic leishmaniosis but also indicate the circulation of L. major and/or L. major/L. donovani s.l. hybrid parasites in Portugal. Also, whenever sequencing of whole Leishmania genomes regularly cannot be accomplished, and while their complete genomes remain under-represented in the nucleotide sequence databases, the combined use of multiple genetic markers, including kinetoplast maxicircle DNA, seems to be essential for typing of Leishmania parasites.

Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle.

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the Leishmania genus. In Australia, leishmaniasis is an imported disease that is presenting itself at increased rates because of international travel, the influx of immigrants, and deployment of military operations to endemic regions. Although Leishmania species are morphologically indistinguishable, there is a strong correlation between some causative species of leishmaniasis and the subsequent response to the treatments available and patient outcome. Consequently, identification of the infective species is imperative as misidentification can result in the administering of an ineffective drug. The aim of this study was to develop a simple diagnostic tool with high sensitivity and specificity, which is capable of detecting the presence of the parasite and accurately differentiating the causative species in question. Using the advantageous properties of the maxi-circle kinetoplast DNA, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) targeting the ND7 gene was developed for the analysis of imported cases of human leishmaniasis in Australia. Designed as a dual analysis, concurrent PCR of Leishmania maxi-circle DNA and digestion with two separate enzymes (NlaIII and HpyCH4IV), this study provides an appraisal on 24 imported cases of leishmaniasis between 2008 and 2017. Five Leishmania species were reported, with members of the Viannia subgenus being the most common. The implementation of novel diagnostic procedures for leishmaniasis such as the one reported here is needed to establish a gold standard practice for the diagnosis and treatment of leishmaniasis.

Analysis of the mitochondrial maxicircle of Trypanosoma lewisi, a neglected human pathogen.

BackgroundThe haemoflagellate Trypanosoma lewisi is a kinetoplastid parasite which, as it has been recently reported to cause human disease, deserves increased attention. Characteristic features of all kinetoplastid flagellates are a uniquely structured mitochondrial DNA or kinetoplast, comprised of a network of catenated DNA circles, and RNA editing of mitochondrial transcripts. The aim of this study was to describe the kinetoplast DNA of T. lewisi.Methods/ResultsIn this study, purified kinetoplast DNA from T. lewisi was sequenced using high-throughput sequencing in combination with sequencing of PCR amplicons. This allowed the assembly of the T. lewisi kinetoplast maxicircle DNA, which is a homologue of the mitochondrial genome in other eukaryotes. The assembly of 23,745 bp comprises the non-coding and coding regions. Comparative analysis of the maxicircle sequence of T. lewisi with Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma brucei and Leishmania tarentolae revealed that it shares 78 %, 77 %, 74 % and 66 % sequence identity with these parasites, respectively. The high GC content in at least 9 maxicircle genes of T. lewisi (ATPase6; NADH dehydrogenase subunits ND3, ND7, ND8 and ND9; G-rich regions GR3 and GR4; cytochrome oxidase subunit COIII and ribosomal protein RPS12) implies that their products may be extensively edited. A detailed analysis of the non-coding region revealed that it contains numerous repeat motifs and palindromes.ConclusionsWe have sequenced and comprehensively annotated the kinetoplast maxicircle of T. lewisi. Our analysis reveals that T. lewisi is closely related to T. cruzi and T. brucei, and may share similar RNA editing patterns with them rather than with L. tarentolae. These findings provide novel insight into the biological features of this emerging human pathogen.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1281-8) contains supplementary material, which is available to authorized users.

Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.

BackgroundLivestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum is widely distributed throughout the world and constitutes an important limitation for the production of animal protein. T. evansi and T. equiperdum are morphologically indistinguishable parasites that evolved from a common ancestor but acquired important biological differences, including host range, mode of transmission, distribution, clinical symptoms and pathogenicity. At a molecular level, T. evansi is characterized by the complete loss of the maxicircles of the kinetoplastic DNA, while T. equiperdum has retained maxicircle fragments similar to those present in T. brucei. T. evansi causes the disease known as Surra, Derrengadera or "mal de cadeiras", while T. equiperdum is the etiological agent of dourine or "mal du coit", characterized by venereal transmission and white patches in the genitalia.MethodsNine Venezuelan Trypanosoma spp. isolates, from horse, donkey or capybara were genotyped and classified using microsatellite analyses and maxicircle genes. The variables from the microsatellite data and the Procyclin PE repeats matrices were combined using the Hill-Smith method and compared to a group of T. evansi, T. equiperdum and T. brucei reference strains from South America, Asia and Africa using Coinertia analysis. Four maxicircle genes (cytb, cox1, a6 and nd8) were amplified by PCRfrom TeAp-N/D1 and TeGu-N/D1, the two Venezuelan isolates that grouped with the T. equiperdum STIB841/OVI strain. These maxicircle sequences were analyzed by nucleotide BLAST and aligned toorthologous genes from the Trypanozoon subgenus by MUSCLE tools. Phylogenetic trees were constructed using Maximum Parsimony (MP) and Maximum Likelihood (ML) with the MEGA5.1® software.ResultsWe characterized microsatellite markers and Procyclin PE repeats of nine Venezuelan Trypanosoma spp. isolates with various degrees of virulence in a mouse model, and compared them to a panel of T. evansi and T. equiperdum reference strains. Coinertia analysis of the combined repeats and previously reported T. brucei brucei microsatellite genotypes revealed three distinct groups. Seven of the Venezuelan isolates grouped with globally distributed T. evansi strains, while TeAp-N/D1 and TeGu-N/D1 strains clustered in a separate group with the T. equiperdum STIB841/OVI strain isolated in South Africa. A third group included T. brucei brucei, two strains previously classified as T. evansi (GX and TC) and one as T. equiperdum (BoTat-1.1). Four maxicircle genes, Cytochrome b, Cythocrome Oxidase subunit 1, ATP synthase subunit 6 and NADH dehydrogenase subunit 8, were identified in the two Venezuelan strains clustering with the T. equiperdum STIB841/OVI strain. Phylogenetic analysis of the cox1 gene sequences further separated these two Venezuelan T. equiperdum strains: TeAp-N/D1 grouped with T. equiperdum strain STIB818 and T. brucei brucei, and TeGu-N/D1 with the T. equiperdum STIB841/OVI strain.ConclusionBased on the Coinertia analysis and maxicircle gene sequence phylogeny, TeAp-N/D1 and TeGu-N/D1 constitute the first confirmed T. equiperdum strains described from Latin America.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1129-2) contains supplementary material, which is available to authorized users.

Identification of the mitochondrially encoded subunit 6 of F1FO ATPase in Trypanosoma brucei

Kinetoplast maxicircle DNA of trypanosomatids encodes eighteen proteins. RNA editing is required to confer translatability to mRNA for twelve of these. Sequence conservation of the predicted hydrophobic polypeptides indicates that they represent functional components of the respiratory chain. Yet, so far only two of those, cytochrome c oxidase subunit I and apocytochrome b of cytochrome c reductase, have been identified with biochemical methods. Here we report on identification of A6 subunit of F1FO ATPase encoded by a pan-edited mRNA in Trypanosoma brucei. The polypeptide was present among the 35S-labeled mitochondrial translation products characterized by anomalous migration in denaturing 2D gels. It was identified as an ATPase subunit by co-migration with this complex in Blue Native 2D gels. A partial N-terminal sequence of the corresponding polypeptide present in the gel-purified ATPase complex from Leishmania tarentolae was consistent with the predicted A6 sequence.

The most unlikely places.

The most unlikely places.

Involvement of a type 2 ribonuclease H in kinetoplast maxicircle DNA replication in Trypanosoma brucei (LB165)

Ribonuclease H (RNase H, EC # 3.1.26.4) catalyzes the hydrolysis of RNA from a DNA/RNA hybrid. This ubiquitous enzyme interacts with several different DNA‐modifying enzymes in the cell and participates in the replication, recombination and repair of nuclear and mitochondrial DNA molecules. We report here that the type 2 RNase H of the parasitic protozoan Trypanosoma brucei is a predominantly mitochondrial enzyme and is essential for the survival of both the procyclic and the blood stream forms. Yeast 2‐hybrid and co‐IP analyses for the mitochondrial DNA replication‐associated proteins interacting directly with RNase HIIC revealed binding of RNase HIIC with the mitochondrial DNA ligase Kα which is involved in kinetoplast DNA replication. Interestingly, we found that RNase HIIC also binds with the DNA recombinase DMC1 which is also located in the mitochondrion of the parasite. Chromatin immunoprecipitation data indicated binding of both RNase HIIC and DMC1 to the maxicircle DNA. We found that the maxicircle DNA and transcripts derived from it are decreased significantly in the cells with decreased levels of RNase HIIC. This enzyme is required for the recovery from DNA damage caused by caffeine, hydroxyurea and methylmethane sulfonate, as the RNase HIIC knocked down cells are more sensitive to the lethality of these DNA damaging agents. We conclude that RNase HIIC collaborates with DNA ligase Kα and DMC1 to participate in the replication and repair of mitochondrial maxicircle DNA in these parasite cells. This research was supported in parts by NIAID grants 5R01AI042327, 1R21AI076757‐01A1 and NIGMS grant #3 S06 GM008037‐34‐S2 to GC.

RNA editing and mitochondrial activity in promastigotes and amastigotes of Leishmania donovani

Kinetoplast maxicircle DNA sequence organisation was investigated in Leishmania donovani, strain 1S LdBob. Gene arrangement in the coding (conserved) region of the maxicircle is collinear with that of most trypanosomatids, with individual genes showing 80–90% nucleotide identity to Leishmania tarentolae, strain UC. The notable exception was an integration of a full-size minicircle sequence in the ND1 gene coding region found in L. donovani. Editing patterns of the mitochondrial mRNAs investigated also followed L. tarentolae UC patterns, including productive editing of the components of respiratory complexes III–V, and ribosomal protein S12 (RPS12), as well as the lack of productive editing in five out of six pan-edited cryptogenes (ND3, ND8, ND9, G3, G4) found in these species. Several guide RNAs for the editing events were localised in minicircles and maxicircles in the locations that are conserved between the species. Mitochondrial activity, including rates of oxygen consumption, the presence and the levels of respiratory complexes and their individual subunits and the steady-state levels of several mitochondrial-encoded mRNAs were essentially the same in axenically grown amastigotes and in promastigotes of L. donovani. However, some modulation of mitochondrial activity between these developmental stages was suggested by the finding of an amastigote-specific component in complex IV, a down-regulation of mitochondrial RNA-binding proteins (MRP) and MRP-associated protein (MRP-AP) in amastigotes, and by variations in the levels of RPS12, ND3, ND9, G3 and G4 pre-edited transcripts.

Mechanism of genetic exchange in American trypanosomes.

The kinetoplastid Protozoa are responsible for devastating diseases. In the Americas, Trypanosoma cruzi is the agent of Chagas' disease--a widespread disease transmissible from animals to humans (zoonosis)--which is transmitted by exposure to infected faeces of blood-sucking triatomine bugs. The presence of genetic exchange in T. cruzi and in Leishmania is much debated. Here, by producing hybrid clones, we show that T. cruzi has an extant capacity for genetic exchange. The mechanism is unusual and distinct from that proposed for the African trypanosome, Trypanosoma brucei. Two biological clones of T. cruzi were transfected to carry different drug-resistance markers, and were passaged together through the entire life cycle. Six double-drug-resistant progeny clones, recovered from the mammalian stage of the life cycle, show fusion of parental genotypes, loss of alleles, homologous recombination, and uniparental inheritance of kinetoplast maxicircle DNA. There are strong genetic parallels between these experimental hybrids and the genotypes among natural isolates of T. cruzi. In this instance, aneuploidy through nuclear hybridization results in recombination across far greater genetic distances than mendelian genetic exchange. This mechanism also parallels genome duplication.

Editing and misediting of transcripts of the kinetoplast maxicircle G5 (ND3) cryptogene in an old laboratory strain of Leishmania tarentolae

RNA editing in the mitochondrion (kinetoplas0 of trypanosomatid protozoa involves a post-transcriptional addition and deletion of uridilate residues [14]. The sites of editing are specified by small guide RNA transcripts (gRNAs), which are encoded in both kinetoplast DNA minicircles and maxicircles. The process initiates with a short region of base-pairing (anchor formation) between a specific gRNA and a pre-edited mRNA, and generally proceeds in a 5' direction with the insertion and deletion of uridilate (U) residues. The precise sites and number of Us inserted and/or deleted is determined by base-pairing with guide adenine or guanine nucleotides in the gRNA. Two basic models have been proposed for the mechanism of this process, one involving two

The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA

The nucleotide sequence of two non-contiguous DNA fragments of 4.0 and 2.2 kb, respectively, of the kinetoplast maxicircle of Trypanosoma brucei brucei EATRO strain 427 has been determined, completing the sequence analysis of the so-called variable region (see also de Vries et al., 1988, Mol. Biochem. Parasitol. 27, 71–82). Analysis of the entire 8-kb variable region sequence revealed the presence of a 5.2-kb cluster of imperfect, tandemly repeated sequences, flanked by DNA of unique sequence. Both repetitive and unique DNA evolve rapidly, but comparison to the closely related strain EATRO 164 indicated that the repetitive cluster is more prone to sequence and size divergence. The variable region is transcribed into RNAs of varying lengths but appears to be devoid of genes encoding mitochondrial proteins or tRNAs, as judged from computer analysis. Moreover, genes that could encode guide RNAs involved in producing the known edited mitochondrial mRNA sequences are also absent. The repetitive DNA cluster within this region consists of 14 blocks each containing one 130 bp repeat and a variable number of 19 bp repeats. A duplicated sequence was identified (5′-GGGGTTGGTGT) which proved to be identical to the eleven 5′-terminal residues of the universal minicircle dodecamer involved in initiation of leading strand synthesis. This suggests a role for these sequences in the initiation of maxicircle DNA replication. With the data presented in this report, the nucleotide sequence analysis of the 23016 bp maxicircle of T. brucei brucei EATRO strain 427 has been completed.

Structure and organization of Marchantia olymorpha chloroplast genome : IV. Inverted repeat and small single copy regions

We characterized the genes in the regions of large inverted repeats (IR A and IR B, 10,058 base-pairs each) and a small single copy (SSC 19,813 bp) of chloroplast DNA from Marchantia polymorpha. The inverted repeat (IR) regions contain genes for four ribosomal RNAs (16 S, 23 S, 4.5 S and 5 S rRNAs) and five transfer RNAs (valine tRNA GAC, isoleucine tRNA GAU, alanine tRNA UGC, arginine tRNA ACG and asparagine tRNA GUU). The gene organization of the IR regions in the liverwort chloroplast genome is conserved, although the IR regions are smaller (10,058 base-pairs) than any reported in higher plant chloroplasts. The small single-copy region (19,813 base-pairs) encoded genes for 17 open reading frames, a leucine tRNA UAG and a proline tRNA GGG-like sequence. We identified 12 open reading frames by homology of their coding sequences to a 4Fe-4S-type ferredoxin protein, a bacterial nitrogenase reductase component (Fe-protein), five human mitochondrial components of NADH dehydrogenase (ND1, ND4, ND4L, ND5 and ND6), two Escherichia coli ribosomal proteins (S15 and L21), two putative proteins encoded in the kinetoplast maxicircle DNA of Leishmania tarentolae (LtORF 3 and LtORF 4), and a bacterial permease inner membrane component (encoded by malF in E. coli or hisQ in Salmonella typhimurium).

The variable region of the Trypanosoma brucei kinetoplast maxicircle: sequence and transcript analysis of a repetitive and a non-repetitive fragment

The sequence of two fragments derived from the variable region of the kinetoplast maxicircle of Trypanosoma brucei has been determined. One fragment (1334 nucleotides, situated immediately upstream of the 12S and 9S ribosomal RNA genes) consists of non-repetitious DNA, which does not hybridize to other maxicircle regions. The other (844 nt, located between 1.7 and 2.55 kb downstream of the NADH-dehydrogenase subunit 5 gene) contains arrays of repetitive sequences which are also found outside this area. Hybridization analysis suggests that approximately 60% of the remaining part of the divergent region, which has not yet been fully analyzed, consists of similar sequences. Neither segment contains genes for mitochondrial proteins or tRNAs, as judged from computer analysis. This conclusion is supported by the fact that maxicircle DNA of trypanosome species other than T. brucei does not cross-hybridize to either fragment. Northern blot analysis and S1 nuclease experiments demonstrate, however, that both maxicircle regions are transcribed into RNAs of varying length (100–3000 nt), albeit at a low level. The function of these transcripts, that are derived from both DNA strands, and the likely absence of protein and tRNA genes from the variable region of the T. brucei maxicircle is discussed.

A minimal ribosomal RNA: sequence and secondary structure of the 9S kinetoplast ribosomal RNA from Leishmania tarentolae.

The portion of the Leishmania tarentolae kinetoplast maxicircle DNA encoding the 9S RNA gene was sequenced, and the 5' and 3' ends of the transcript were determined. A secondary structure for the 9S RNA was determined based on the Escherichia coli 16S model. The 610-nucleotide 9S RNA exhibits a minimal secondary structure in which all four domains of the E. coli 16S structure are preserved. Within domains, however, some stems and loops have been greatly reduced or eliminated entirely. It is presumed that these reduced domains represent the minimal essential small ribosomal RNA secondary structures necessary for a functional ribosome. Alignment of the L. tarentolae 9S rRNA sequence with the published Trypanosoma brucei 9S rRNA sequence shows a nucleotide similarity of 84% and a transversion/transition ratio of 1.66.

The divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA contains a diverse set of repetitive sequences.

A 2.76 kb segment of the 12 kb divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA consists almost entirely of repeated sequences. The repeats can be grouped into six families, some of which are present throughout the remainder of the divergent region. The repeats are oriented in a head-to-tail fashion with the three simplest repeats clustered into large arrays. A 47 bp palindrome and two copies of a "supercluster" of three different types of repeats are also present in the sequenced region. A sequence change in the divergent region is described for a clonal strain of L. tarentolae which was passaged continuously for several years. The repetitive sequences found in the divergent region appear to be appropriate substrates for the presumed deletion/insertion/recombination events occurring in this rapidly evolving portion of the maxicircle.

Sequences of six genes and several open reading frames in the kinetoplast maxicircle DNA of Leishmania tarentolae.

The DNA sequence of approximately 80% of the transcribed region of the kinetoplast maxicircle DNA of Leishmania tarentolae was obtained, and structural genes were localized by comparison of the translated amino acid sequences with those of known mitochondrial genes from other organisms. By this method, the genes for cytochrome oxidase subunits I, II, and III, cytochrome b, and human mitochondrial unidentified reading frames 4 and 5 were identified. By comparing the amino acid sequences of the putative L. tarentolae genes with those of known genes, we conclude that TGA codes for tryptophan, as in most other mitochondrial systems. This is the only apparent change from the universal genetic code. The six identified structural genes show various degrees of divergence from the homologous genes in other species, with cytochrome oxidase subunit I being the most conserved and cytochrome oxidase subunit III being the least conserved. A comparison of the cytochrome b genes from L. tarentolae and Trypanosoma brucei showed that the ratio of transversions to transitions is 1:1, suggesting that these species diverged from each other more than 80 X 10(6) years ago. Several as yet unidentified open reading frames were also present in the maxicircle sequence. These data confirm that maxicircle DNA has a coding potential which typifies other mitochondrial systems.

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 17 kbp 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.