Minicircle Kinetoplast Genome of Insect Trypanosomatid Leptomonas pyrrhocoris.

We present here the structure of a minicircle population based on transcriptome sequencing of Leptomonas pyrrhocoris. We show that minicircle DNA molecules are dimeric. As in dixenous species, the entire molecule of minicircle DNA is transcribed. This is the first minicircle transcriptome of monoxenous trypanosomatid species determined using NGS technology.

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.

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.

[Three patterns of trypanosomatid cryptogene structural organization].

We sequenced a number of cryptogenes from previously unstudied species of homoxenous trypanosomatids belonging to the different phylogenetic groups and found new examples of editing domain length reduction for A6 and COIII. The comparative analyzes of sequences allows to divide the cryptogenes in three groups (patterns) according to the degree of primary structure conservation and editing domain length variation. We discuss the possible factors which influence the cryptogene's structure and evolutionary behavior. Also we demonstrate alternative editing of rps12 transcript in Wallaceina sp. Wsd.

[The epidemiological features of visceral leishmaniasis, revealed on examination of children by polymerase chain reaction, in the Papsky District, Namangan Region, Uzbekistan].

Patients with visceral leishmaniasis (VL) have been registered in the Papsky District, Namangan Region, Uzbekistan, over the past 23 years. A total of 95 patients were notified in 1987 to 2009. In 2007-2008, a mass survey using the polymerase chain reaction (PCR) within the international INTAS project 05-100006-8043 was conducted in 5 population aggregates of the Papsky District, Namangan Region, Uzbekistan, where VL cases had been regularly registered in the last years. Bone marrow and venous and peripheral blood smears were used as a test material. A total of 234 samples, including 3 bone marrow biopsy specimens, 9 venous blood samples and 222 peripheral blood ones, were tested. All the samples were on the glass slides. Three groups were identified among the examinees. Group 1 consisted of 13 subjects who had been ill at different times. Group 2 comprised 27 children treated at hospital for various diagnoses. Group 3 (the largest one, n=190) included apparently healthy children. All the children of this group felt well and had no symptoms of any illnesses at the examination. In this group, 85 (44.7%) subjects were PCR-positive. Twenty-four (55.8%) of 43 children in the age group of 0-3 years were PCR-positive; the 4-7-year age group comprised 66 subjects and 33 (50%) of them were PCR-positive. Group over 7 years of age included 81 subjects; 45 (55.5%) were PCR-positive. The results of the mass survey with PCR, which covered the 5 population aggregates in the Papsky District, Namangan Region, Uzbekistan, suggest the epidemic activity of a synathropic focus of VL and make us look at many fixed notions of its epidemiology in new contexts.

[Monitoring the spread of the SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) variants in the Moscow region using targeted high-throughput sequencing].

INTRODUCTION: Since the outbreak of the COVID-19 pandemic caused by SARS-CoV-2 novel coronavirus, the international community has been concerned about the emergence of mutations altering some biological properties of the pathogen like increasing its infectivity or virulence. Particularly, since the end of 2020, several variants of concern have been identified around the world, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). However, the existing mechanism of detecting important mutations are not always effective enough, since only a relatively small part of all pathogen samples can be examined by whole genome sequencing due to its high cost. MATERIAL AND METHODS: In this study, we have designed special primer panel and used it for targeted highthroughput sequencing of several significant S-gene (spike) regions of SARS-CoV-2. The Illumina platform averaged approximately 50,000 paired-end reads with a length of ≥150 bp per sample. This method was used to examine 579 random samples obtained from COVID-19 patients in Moscow and the Moscow region from February to June 2021. RESULTS: This study demonstrated the dynamics of distribution of several SARS-CoV-2 strains and its some single mutations. It was found that the Delta strain appeared in the region in May 2021, and became prevalent in June, partially displacing other strains. DISCUSSION: The obtained results provide an opportunity to assign the viral samples to one of the strains, including the previously mentioned in time- and cost-effective manner. The approach can be used for standardization of the procedure of searching for mutations in individual regions of the SARS-CoV-2 genome. It allows to get a more detailed data about the epidemiological situation in a region.

[Screening of population genetic SCAR markers for mykiss Parasalmo (Oncorhynchus) mykiss from Kamchatka].

Using AP-PCR, the genome of Kamchatka mykiss (Parasalmo (O.) mykiss) was examined. Polymorphic fragments, implying geographic differences among the samples, were selected, cloned, and sequenced. Based on these sequences, longer, specific SCAR primers were selected and constructed. Using the BLAST software program, the sequences were analyzed for analogy to those from the GenBank database. It seemed likely that all sequences obtained belonged to earlier unexamined repeated sequences, variable in the populations of the species of interest. A total of seven SCAR markers, characterized by population-significant variability of the DNA products in Kamchatka geographic group of rainbow trout were constructed. These markers can be used for further investigation of the species Parasalmo (O.) mykiss. The SCAR marker sequences were deposited in GenBank under the accession numbers EU805500 to EU805506.

[Genetic polymorphism of Commander Islands polar foxes Alopex lagopus semenovi Ognev, 1931 and Alopex lagopus beringiensis Merriam, 1902].

The polymorphism of the mitochondrial DNA (mtDNA) control region sequence was examined in 30 polar foxes from Bering Island and 30 polar foxes from Mednyi Island. Seven haplotypes were revealed in polar foxes from Bering Island, and one, in polar foxes from Mednyi Island. The age of divergence of these populations (12 000 +/- 600 years) was calculated based on a fragment of the D-loop. In Bering polar foxes, the sequence nucleotide diversity (pi) was 0.003 (S.D. = 0.002), the haplotype diversity h in Bering polar foxes was 0.835 (S.D. = 0.037). The effective number of females n the Bering Island population was estimated as 105 animals.

[ATPase subunit 6 gene of Leptomonas seymouri (Trypanosomatidae) is transcribed and edited as a polycistronic transcript].

The maturation of mitochondrial RNA transcripts proceeds through several steps. Here we use insects trypanosomatid Leptomonas seymouri as a model organism for analysis of transcription and posttranscriptional processing of mitochondria encoded gene for the subunit 6 of ATPase (A6). It was shown that Cyt b (cytochrome b) and A6 genes were transcribed and edited as a polycistronic template. Analysis of twelve RT-PCR products of both genes led to identification of four types of differently and/or partially edited cDNA molecules. Based on the analysis of two fully edited A6 transcripts we propose the existence of two alternatively edited products.

Structural organization of kinetoplast DNA and its compaction in the in vitro model system.

Electron microscopic studies of Leishmania gymnodactyli cells lysed at hypotonic conditions showed that the structures identified as kinetoplast DNA have the appearance of loose accumulations of crossed and sometimes branched rod-like structures 100 to 200 nm long and 20 nm thick. The compaction of isolated kinetoplast DNA (kpDNA) caused by interaction with synthetic tripeptide--dansylhydrazide trivaline--was also studied. The analysis of the structures arising at different steps of compaction showed that the minicircles are compacted forming rod-like structures where minicircle double-stranded DNA segments are closely associated side by side in a manner which was earlier described for initial compaction stages of "triple rings". These rod-like structures resemble in their appearance the structures found in lysed cell preparations obtained according to Miller's method. Branching of rod-like structures can be the consequence of minicircle catenation. In vitro compaction is completed with the formation of a compacted network, its diameter being 3 to 6 times smaller as compared with the initial one.

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' noncoding) 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.

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.

Characterization of kinetoplast minicircle DNA in the lower trypanosomatid Crithidia oncopelti.

The kinetoplast DNA of the lower trypanosomatid Crithidia oncopelti contains minicircles of four major size classes. Attempts to clone these molecules revealed the unclonability of the full-length minicircles in pUC and M13 vectors. A few clones were obtained using a lambda insertion vector lambda XIII. The nucleotide sequence of a cloned, apparently full-length minicircle has been determined and characterized. This 1848-bp molecule contains a single region corresponding to the 'conserved' minicircle region in other trypanosomatid species and an area with some features of the bent helix. About one-third of the molecule length is occupied by an extremely (T+G) versus (A+C) strand biased region. The role of this region, as well as the significance of the ORFs found and the putative secondary structures potentially formed by the minicircle sequence, remains unknown. The hybridization of kDNA with the different regions of a cloned minicircle shows that there are some segments of DNA specific to a particular class or a group of classes. Such a mode of sequence heterogeneity suggests that the possible functions (if any) of different minicircle classes may differ.

Genetic heterogeneity in the species Leishmania tropica revealed by different PCR-based methods.

A PCR fingerprinting approach, using single non-specific primers, as well as restriction and single-strand conformation polymorphism (SSCP) analyses of the amplified ribosomal internal transcribed spacer, were used to investigate genetic variability in the species Leishmania tropica. Twenty-nine strains of the 'L. tropica complex' from different Old World geographical areas were studied including 4 from Namibia, and 1 strain of L. killicki. All techniques revealed a high degree of genetic heterogeneity among the strains of L. tropica. The PCR fingerprinting displayed the highest discriminatory power, but can be applied only to cultured parasites. The internal transcribed spacer (ITS) region can be amplified directly from infected clinical samples and analysed subsequently. No strict correlation was discerned between the genetic variants and either the geographical origin of the strains or the clinical manifestations associated with human disease, except for the Namibian strains. Also, genetic variation did not correlate well with characterization by enzyme variant electrophoretic analysis. The strain of L. killicki always clustered together with the strains of L. tropica, suggesting it, probably, should not be considered a separate species of Leishmania. However, the 4 Namibian strains formed a distinct, statistically well-supported group closely related to but different from the other strains of L. tropica.

Atomic force and electron microscopy of high molecular weight circular DNA complexes with synthetic oligopeptide trivaline.

Intramolecular compact structures formed by high molecular weight circular superhelical DNA molecules due to interaction with synthetic oligopeptide trivaline (1) were studied by atomic force and electron microscopy. Three DNA preparations were used: plasmids pTbol, pRX10 and cosmid 27,877, with sizes 6,120 bp, 10,500 bp and 44,890 bp respectively. Plasmid pTbo1 and pRX10 preparations along with monomers contained significant amount of dimers and trimers. Main structures in all preparations observed were compact particles, which coincide in their appearance and compaction coefficient (3,5-3,7) with triple rings described earlier. The size and structure characteristics of triple rings and other compact particles on atomic force images in general coincide with those obtained by EM (2). AFM (3) images allow to get additional information about the ultrastructural organization and arrangement of DNA fibers within the compact structures. Along with triple rings in pTbol and pRX10-TVP complexes significant amount of compact structures were observed having the shape of two or three compact rings attached to each other by a region of compact fibre. Basing on the data of contour length measurements and the shape of the particles it was concluded that these structures were formed due to compaction of dimeric and trimeric circular DNA molecules. Structures consisting of several attached to each other triple rings were not found for pTbol, pRX10 monomers or cosmid preparations--TVP complexes where only single triple rings were observed. The conclusion is made that initiation of compact fibre formation within the circular molecules depends on the primary structure and for dimeric or trimeric circular molecules two or three compaction initiation points are present, located in each monomer unit within one circular DNA molecule. The nucleotide sequence dependent compaction mechanism providing independent compaction of portions of one circular molecule can be of interest for understanding of DNA compaction processes in vivo.

Phylogenetic analysis of Trypanosomatina (Protozoa: Kinetoplastida) based on minicircle conserved regions.

Phylogenetic relationships within the suborder Trypanosomatina were inferred from the kinetoplast DNA minicircle conserved region sequences. Trees built using distance-matrix (Neighbor-Joining) and maximum parsimony methods showed that the minicircle conserved regions (CRs) provide a sensitive and specific molecular marker suitable for phylogenetic analyses of subspecies and strains of trypanosomatid flagellates, as testified by the subdivision of the genus Leishmania into the subgenera Leishmania. Viannia and Sauroleishmania. However, since Phytomonas and monogenetic parasites of insects represent the earliest diverging groups, the CRs do not seem to be useful for inference of relationships among major lineages of the order Kinetoplastida.

Analysis of kinetoplast DNA of freshwater fish trypanosomes.

The sequences of 10 conservative regions (CR) of minicircles of 6 selected isolates of freshwater fish trypanosomes have typical organization of this region with high degree of sequence conservation. The comparison with CRs of other trypanosomatids showed that freshwater fish trypanosomes represent a compact separate group within the genus Trypanosoma. The alignment of all sequences obtained revealed, however, the existence of 2 types of CRs in sequenced minicircles, with the differences concentrated in a short region. Taxonomic consequences of these results are discussed.

[Minicircular kinetoplast DNA from Trypanosomatidae]. / Minikol'tsevaia kinetoplastnaia DNK 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.

[Various elements of the structure of mini-ring kinetoplast DNA of Crithidia fasciculata]. / Nekotorye élementy struktury mini-kol'tsevoi kinetoplastnoi DNK Crithidia fasciculata.

The nucleotide sequence of 1.4 kbp SmaI-fragment of minicircle DNA from kinetoplasts of Crithidia fasciculata has been determined and some sequence elements characterized. The sequence contains several oligo(dT)blocks located on the same strand in phase with a period of DNA helix turn, thus representing a "bent helix". Both sides of the bent helix region are flanked by sequences capable of forming a cloverleaf structure. There are also two direct 150 bp repeats located 180 degrees apart on the circular map of the molecule. Each repeat contains the sites of H-strand and L-strand replication origin. The specific stem-loop secondary structure may be folded by the nucleotide sequence within the origins region. The alignment of the sequence determined with two other C. fasciculata minicircle sequences spanning over the bent helix and the adjacent regions has indicated the presence of several conserved sequence blocks, one of them representing the sequence of the bend. The divergence of three sequences occurred mainly by small insertions-deletions. Several open reading frames were found, the largest of which being capable of coding for the approximately 200 amino acids polypeptide.

[The identification of 12S rRNA gene promotor in Leptomonas seymouri mitochondrial DNA]. / Identifikatsiia promotora gena 12S rRNK v mitokhondrial'noi DNK Leptomonas seymouri.

The fragment of kinetoplast maxicircle (mitochondrial) DNA divergent region Leptomonas seymouri was localized containing promotor of 12S rRNA gene. The ability of this fragment to initiate transcription was tested with transcription system in organello in isolated mitochondria of L. seymouri.