The mitochondrial genome of Dendrobaena tellermanica Perel, 1966 (Annelida: Lumbricidae) and its phylogenetic position.

Earthworms are an important ecological group that has a significant impact on soil fauna as well as plant communities. Despite their importance, genetic diversity and phylogeny of earthworms are still insufficiently studied. Most studies on earthworm genetic diversity are currently based on a few mitochondrial and nuclear genes. Mitochondrial genomes are becoming a promising target for phylogeny reconstruction in earthworms. However, most studies on earthworm mitochondrial genomes were made on West European and East Asian species, with much less sampling from other regions. In this study, we performed sequencing, assembly, and analysis of the mitochondrial genome of Dendrobaena tellermanica Perel, 1966 from the Northern Caucasus. This species was earlier included into D. schmidti (Michaelsen, 1907), a polytypic species with many subspecies. The genome was assembled as a single contig 15,298 bp long which contained a typical gene set: 13 protein-coding genes (three subunits of cytochrome c oxidase, seven subunits of NADH dehydrogenase, two subunits of ATP synthetase, and cytochrome b), 12S and 16S ribosomal RNA genes, and 22 tRNA genes. All genes were located on one DNA strand. The assembled part of the control region, located between the tRNA-Arg and tRNA-His genes, was 727 bp long. The control region contained multiple hairpins, as well as tandem repeats of the AACGCTT monomer. Phylogenetic analysis based on the complete mitochondrial genomes indicated that the genus Dendrobaena occupied the basal position within Lumbricidae. D. tellermanica was a rather distant relative of the cosmopolitan D. octaedra, suggesting high genetic diversity in this genus. D. schmidti turned out to be paraphyletic with respect to D. tellermanica. Since D. schmidti is known to contain very high genetic diversity, these results may indicate that it may be split into several species.

De novo assembly and analysis of the transcriptome of the Siberian wood frog Rana amurensis.

The Siberian wood frog Rana amurensis Boulenger, 1886 is the most hypoxia-tolerant amphibian. It can survive for several months in an almost complete absence of oxygen. Little is known about the mechanisms of this remarkable resilience, in part because studies of amphibian genomes are impeded by their large size. To make the Siberian wood frog more amenable for genetic analysis, we performed transcriptome sequencing and de novo assembly for the R. amurensis brain under hypoxia and normoxia, as well as for the normoxic heart. In order to build a de novo transcriptome assembly of R. amurensis, we utilized 125-bp paired-end reads obtained from the brain under normoxia and hypoxia conditions, and from the heart under normoxia. In the transcriptome assembled from about 100,000,000 reads, 81.5 % of transcripts were annotated as complete, 5.3 % as fragmented, and 13.2 % as missing. We detected 59,078 known transcripts that clustered into 22,251 genes; 11,482 of them were assigned to specific GO categories. Among them, we found 6696 genes involved in protein binding, 3531 genes involved in catalytic activity, and 576 genes associated with transporter activity. A search for genes encoding receptors of the most important neurotransmitters, which may participate in the response to hypoxia, resulted in a set of expressed receptors of dopamine, serotonin, GABA, glutamate, acetylcholine, and norepinephrine. Unexpectedly, no transcripts for histamine receptors were found. The data obtained in this study create a valuable resource for studying the mechanisms of hypoxia tolerance in the Siberian wood frog, as well as for amphibian studies in general.

Variation in nuclear genome size within the Eisenia nordenskioldi complex (Lumbricidae, Annelida).

The size of the nuclear genome in eukaryotes is mostly determined by mobile elements and noncoding sequences and may vary within wide limits. It can differ signif icantly both among higher-order taxa and closely related species within a genus; genome size is known to be uncorrelated with organism complexity (the so-called C-paradox). Less is known about intraspecif ic variation of this parameter. Typically, genome size is stable within a species, and the known exceptions turn out be cryptic taxa. The Eisenia nordenskioldi complex encompasses several closely related earthworm species. They are widely distributed in the Urals, Siberia, and the Russian Far East, as well as adjacent regions. This complex is characterized by signif icant morphological, chromosomal, ecological, and genetic variation. The aim of our study was to estimate the nuclear genome size in several genetic lineages of the E. nordenskioldi complex using f low cytometry. The genome size in different genetic lineages differed strongly, which supports the hypothesis that they are separate species. We found two groups of lineages, with small (250-500 Mbp) and large (2300-3500 Mbp) genomes. Moreover, different populations within one lineage also demonstrated variation in genome size (15-25 %). We compared the obtained data to phylogenetic trees based on transcriptome data. Genome size in ancestral population was more likely to be big. It increased or decreased independently in different lineages, and these processes could be associated with changes in genome size and/or transition to endogeic lifestyle.

Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album.

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that - at a subsequent stage of transfection into yeast cells - it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein's secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 µg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

Morphotypes and genetic diversity of Dendrobaena schmidti (Lumbricidae, Annelida).

Dendrobaena schmidti (Michaelsen, 1907) is a polymorphic earthworm species from the Caucasus and adjacent regions. Adult D. schmidti individuals have highly variable body size (from 1.5 to well over 10 cm) and color (from dark purple to total lack of pigmentation), so a lot of subspecies of D. schmidti have been described; however, the existence of most of them is currently under dispute. We studied the genetic diversity of D. schmidti from seven locations from the Western Caucasus using mitochondrial (a fragment of the cytochrome oxidase I gene) and nuclear (internal ribosomal transcribed spacer 2) DNA. For both genes studied, we found that our sample was split into two groups. The first group included somewhat bigger (3-7.5 cm) individuals that were only slightly pigmented or totally unpigmented (when fixed by ethanol). The second group contained small (1.7-3.5 cm) specimens with dark purple pigmentation. In one of the studied locations these two groups were found in sympatry. However, there were no absolute differences either in general appearance (pigmented/unpigmented, small/big) or among diagnostic characters. Although the two groups differed in size (the majority of individuals from the first group were 5-6 cm long, and of the second one, 2-3 cm), the studied samples overlapped to a certain degree. Pigmentation, despite apparent differences, was also unreliable, since it was heavily affected by fixation of the specimens. Thus, based on the obtained data we can conclude that D. schmidti consists of at least two species that have identical states of diagnostic characters, but differ in general appearance.

Erratum to: "Gas chromatography-mass spectrometry in the taxonomy of Miscanthus".

Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2019;23(8):1076-1081 (in Russian) Page 1081, in Acknowledgements instead of This work was supported by State Budgeted Project АААА-А17-117092070032-4. should read This work was supported by State Budgeted Project 0259-2019-0011. The original article can be found under DOI 10.18699/VJ19.583.

Diversity and occurrence of methylotrophic yeasts used in genetic engineering.

Methylotrophic yeasts have been used as the platform for expression of heterologous proteins since the 1980's. They are highly productive and allow producing eukaryotic proteins with an acceptable glycosylation level. The first Pichia pastoris-based system for expression of recombinant protein was developed on the basis of the treeexudate- derived strain obtained in the US southwest. Being distributed free of charge for scientific purposes, this system has become popular around the world. As methylotrophic yeasts were classified in accordance with biomolecular markers, strains used for production of recombinant protein were reclassified as Komagataella phaffii. Although patent legislation suggests free access to these yeasts, they have been distributed on a contract basis. Whereas their status for commercial use is undetermined, the search for alternative stains for expression of recombinant protein continues. Strains of other species of methylotrophic yeasts have been adapted, among which the genus Ogataea representatives prevail. Despite the phylogenetic gap between the genus Ogataea and the genus Komagataella representatives, it turned out possible to use classic vectors and promoters for expression of recombinant protein in all cases. There exist expression systems based on other strains of the genus Komagataella as well as the genus Candida. The potential of these microorganisms for genetic engineering is far from exhausted. Both improvement of existing expression systems and development of new ones on the basis of strains obtained from nature are advantageous. Historically, strains obtained on the southwest of the USA were used as expression systems up to 2009. Currently, expression systems based on strains obtained in Thailand are gaining popularity. Since this group of microorganisms is widely represented around the world both in nature and in urban environments, it may reasonably be expected that new expression systems for recombinant proteins based on strains obtained in other regions of the globe will appear.

An integrated method for taxonomic identification of microorganisms.

For accurate species-level identification of microorganisms, researchers today increasingly use a combination of standard microbiological cultivation and visual observation methods with molecular biological and genetic techniques that help distinguish between species and strains of microorganisms at the level of DNA or RNA molecules. The aim of this work was to identify microorganisms from the ICG SB RAS Collection using an integrated approach that involves a combination of various phenotypic and genotypic characteristics. Key molecular-genetic and phenotypic characteristics were determined for 93 microbial strains from the ICG SB RAS Collection. The strains were characterized by means of morphological, physiological, moleculargenetic, and mass-spectrometric parameters. Specific features of the growth of the strains on different media were determined, and cell morphology was evaluated. The strains were tested for the ability to utilize various substrates. The strains studied were found to significantly differ in their biochemical characteristics. Physiological characteristics of the strains from the collection were identified too, e.g., the relationship with oxygen, type of nutrition, suitable temperature and pH ranges, and NaCl tolerance. In this work, the microorganisms analyzed were combined into separate groups based on the similarities of their phenotypic characteristics. This categorization, after further refinement and expansion of the spectrum of taxa and their metabolic maps, may serve as the basis for the creation of an "artificial" classification that can be used as a key for simplified and quicker identification and recognition of microorganisms within both the ICG SB RAS Collection and other collections.

A novel thermophilic Aeribacillus bacteriophage AP45 isolated from the Valley of Geysers, Kamchatka: genome analysis suggests the existence of a new genus within the Siphoviridae family.

A novel thermophilic bacteriophage AP45 and its host strain Aeribacillus sp. CEMTC656 were isolated from the Valley of Geysers, Kamchatka Peninsula, Russia. Bacteriophage AP45 was identified as a member of the Siphoviridae family by electron microscopy. It showed high thermostability and had a slow cycle of reproduction. The AP45 genome had 51,606 base pairs (bp) and contained 71 open reading frames (ORFs), 40 of them encoding proteins of predicted function. Genes encoding DNA and RNA polymerases were not identified, indicating that AP45 used host polymerases. Based on the ORF65 encoding putative endolysin, the recombinant protein rAP45Lys was developed and its peptidoglycan-hydrolyzing activity was demonstrated. The AP45 genome exhibited limited identity to other phage sequences; the highest identity, 36%, was with the genome of the thermophilic Geobacillus myovirus D6E. The majority of putative proteins encoded by the AP45 genome had higher similarity to proteins from bacteria belonging to the Bacillaceae family, than to bacteriophages. In addition, more than half of the putative ORFs in the AP45 genome were highly similar to prophage sequences of A. pallidus strain 8m3, which was isolated in north-east China. The AP45 phage and revealed prophages might be members of a new genus belonging to the Siphoviridae family.

Proteomic Study of Blood Serum in Coronary Atherosclerosis.

Changes in the blood serum proteins were assessed in men with coronary atherosclerosis and without coronary heart disease. Proteins were separated by 2D-electrophoresis, protein fractions were identified by their peptide fingerprint by MALDI method; fractions with more than twofold increase in protein level were determined. In blood serum of patients with coronary atherosclerosis, the content of C4 complement protein increased and ceruloplasmin level decreased, which is typical of heart failure and coronary heart disease.

[MODERN METHODS OF PHYSICAL-CHEMICAL RESEARCH IN GASTROENTEROLOGY PRACTICE: THE EXPERIENCE OF INTERACTION].

UNLABELLED: The aim of this work was to assess the potential of some physical and chemical methods for studying erythrocytes and blood serum in gastroenterological practice by the example of colorectal cancer (CC). MATERIALS AND METHODS: A total of 26 persons with various stages of colorectal cancer and 16 healthy (control group) were examined. Parameters of erythrocytes and blood serum were investigated by light microscopy, dielectrophoresis in a non-uniform alternating electric field (DEF in NUAEF), terahertz spectroscopy, ellipsometry, Raman-spectroscopy. RESULTS: Polymorphism of erythrocytes, rigidity, viscosity, indexes of aggregation and destruction were significantly higher in patients with CC and polarizability, amplitude of erythrocyte deformation in NUAEF being lower than those in the controls. The study of erythrocytes by terahertz spectroscopy revealed the low levels of amplitude transmittance over the whole frequency range in CC patients compared to the controls. The increasing of refractive index, degree of heterogeneity of thin films obtained from the serum in CC patients were observed in considering the ellipsometric parameters. We found a significant increasing of the concentration of antigens to CD24 at the early stage of the disease. The areas of some peaks in Raman spectra were significantly lower in patients with CC compared to the healthy ones, it is possible due to a carotin deficiency. Most of the studied parameters were correlated with the stage of the disease. A set of optical methods for studying blood serum compared with those of histology and radiological methods of diagnosis showed their high sensitivity and specificity, positive and negative predictive value (80 % and above). CONCLUSION: The obtained results of the pilot study demonstrate the prospects of using physical and chemical methods of research of erythrocytes and blood serum for early diagnosis, stage of disease and monitoring the effectiveness of treatment of CC.

No DNA damage response and negligible genome-wide transcriptional changes in human embryonic stem cells exposed to terahertz radiation.

Terahertz (THz) radiation was proposed recently for use in various applications, including medical imaging and security scanners. However, there are concerns regarding the possible biological effects of non-ionising electromagnetic radiation in the THz range on cells. Human embryonic stem cells (hESCs) are extremely sensitive to environmental stimuli, and we therefore utilised this cell model to investigate the non-thermal effects of THz irradiation. We studied DNA damage and transcriptome responses in hESCs exposed to narrow-band THz radiation (2.3 THz) under strict temperature control. The transcription of approximately 1% of genes was subtly increased following THz irradiation. Functional annotation enrichment analysis of differentially expressed genes revealed 15 functional classes, which were mostly related to mitochondria. Terahertz irradiation did not induce the formation of γH2AX foci or structural chromosomal aberrations in hESCs. We did not observe any effect on the mitotic index or morphology of the hESCs following THz exposure.

Nonlinear electrophoresis of protein-detergent complexes.

A comparatively new procedure is described for the nonlinear electrophoresis of proteins. Movement and separation of complexes formed by proteins and ionic detergents is first experimentally demonstrated for SDS rainbow colored protein molecular weight markers (Amersham). This result was revealed by SDS-PAGE in an asymmetric zero average pulsed electric field with a peak amplitude of up to 300 V cm(-1) and a frequency of 100 Hz. The highest molecular weight fractions were found to have the highest nonlinear drift velocity. A two-dimensional map of distribution of the protein complexes developed using nonlinear electrophoresis followed by SDS gel electrophoresis in an orthogonal direction, reveals nonuniform distribution of the fractions. Nonlinear electrophoresis can be run without electrode chambers, since the buffer electrolyte is not used up in alternating electric fields. Thus, this new type of electrophoresis can have advantages in microfluidic systems and biochips. Also possible uses are discussed of nonlinear electrophoresis via nonlinear focusing of protein-detergent complexes for further improvement of the SDS-PAGE technique for the separation and examination of these large hydrophobic complexes.