[Age Dynamics of Telomere Length in Endemic Baikal Planarians].

Age-related changes in telomere length (TL) in somatic tissues are not limited only to shortening. It is known that many organisms show different TL dynamics. Such species specificity indicates the complexity of the mechanisms involved in the regulation of TL. Owing to their morphological, physiological, and ecological features, Baikal planarians are an interesting model for studying the TL dynamics and the factors influencing it in comparison with species living outside Baikal. In this work, we investigated telomerase activity and age-related changes in TL in three endemic species of planarians from the Dendrocoelidae family. Two species are giant deep-water species (7-12 cm long, Sorocelis hepatizon and Rimacephalus arecepta), and one is a coastal shallow species (1 cm long, Baikalobia guttata). In addition, we investigated the telomere biology in another small Siberian species from the Planariidae family (2 cm in length, Phagocata sibirica), which is not found in Baikal. TL and telomerase activity were determined using real-time PCR and the TRAP method. Three types of age-related TL dynamics were detected with active telomerase: (1) TL shortening at the juvenile stage of development and subsequent maintenance (R. arecepta, Ph. sibirica), (2) gradual TL shortening during ontogeny (S. hepatizon) and (3) cyclic dynamics of TL (B. guttata). Thus, the changes of TL in the studied planarians does not have an obvious connection with body size, habitat depth, phylogenetic relationship and is probably a consequence of species features in the regulation of telomerase activity.

Conserved molecular structure of the centromeric histone CENH3 in Secale and its phylogenetic relationships.

It has been repeatedly demonstrated that the centromere-specific histone H3 (CENH3), a key component of the centromere, shows considerable variability between species within taxa. We determined the molecular structure and phylogenetic relationships of CENH3 in 11 Secale species and subspecies that possess distinct pollination systems and are adapted to a wide range of abiotic and biotic stresses. The rye (Secale cereale) genome encodes two paralogous CENH3 genes, which differ in intron-exon structure and are transcribed into two main forms of the protein, αCENH3 and ßCENH3. These two forms differ in size and amino acid substitutions. In contrast to the reported differences in CENH3 structure between species within other taxa, the main forms of this protein in Secale species and subspecies have a nearly identical structure except some nonsynonymous substitutions. The CENH3 proteins are strictly controlled by genetic factors responsible for purifying selection. A comparison between Hordeum, Secale and Triticum species demonstrates that the structure of CENH3 in the subtribes Hordeinae and Triticinae evolved at different rates. The assumption that reticulate evolution served as a factor stabilizing the structure and evolutionary rate of CENH3 and that this factor was more powerful within Secale and Triticum than in Hordeum, is discussed.

The expansion of heterochromatin blocks in rye reflects the co-amplification of tandem repeats and adjacent transposable elements.

BACKGROUND: A prominent and distinctive feature of the rye (Secale cereale) chromosomes is the presence of massive blocks of subtelomeric heterochromatin, the size of which is correlated with the copy number of tandem arrays. The rapidity with which these regions have formed over the period of speciation remains unexplained. RESULTS: Using a BAC library created from the short arm telosome of rye chromosome 1R we uncovered numerous arrays of the pSc200 and pSc250 tandem repeat families which are concentrated in subtelomeric heterochromatin and identified the adjacent DNA sequences. The arrays show significant heterogeneity in monomer organization. 454 reads were used to gain a representation of the expansion of these tandem repeats across the whole rye genome. The presence of multiple, relatively short monomer arrays, coupled with the mainly star-like topology of the monomer phylogenetic trees, was taken as indicative of a rapid expansion of the pSc200 and pSc250 arrays. The evolution of subtelomeric heterochromatin appears to have included a significant contribution of illegitimate recombination. The composition of transposable elements (TEs) within the regions flanking the pSc200 and pSc250 arrays differed markedly from that in the genome a whole. Solo-LTRs were strongly enriched, suggestive of a history of active ectopic exchange. Several DNA motifs were over-represented within the LTR sequences. CONCLUSION: The large blocks of subtelomeric heterochromatin have arisen from the combined activity of TEs and the expansion of the tandem repeats. The expansion was likely based on a highly complex network of recombination mechanisms.

[The first experience with a new technique of portal endoscopic discectomy for herniated cervical discs]. / Pervyi opyt primeneniya novoi tekhniki portal'noi endoskopicheskoi diskektomii pri gryzhakh mezhpozvonkovykh diskov sheinogo otdela pozvonochnika.

Choosing the most appropriate tactics for surgical treatment of herniated cervical discs is a topical issue to be discussed. The idea of herniated disc removal using an endoscopic technique is not new. This is routine surgery for the lumbar spine. However, application of endoscopic techniques in surgery on the cervical spine was first reported only in 2014 (J. Yang, et al.). OBJECTIVE: To master the methodology of a new technique, portal endoscopic discectomy, and define the indications for this surgery on herniated cervical discs; to compare outcomes of this surgery with outcomes of anterior microsurgical discectomy. MATERIAL AND METHODS: The study included 25 patients who underwent portal endoscopic cervical discectomy. A comparison group consisted of 25 patients who underwent anterior microsurgical discectomy and placement of an interbody cage. RESULTS: A comparison of the results of surgeries revealed no significant difference (p>0.05) in the degree of postoperative local and radicular pain syndrome. According to the Neck Disability Index (NDI), a significant improvement occurred in patients with endoscopic surgery. According to the Odom criterion, a significant advantage in the number of excellent and good outcomes occurred in patients of the study group. There were significant differences between groups in the duration of postoperative hospital stay. The duration was 3 days in the study group and 5 days in the control group, on average. CONCLUSION: Portal endoscopic discectomy is highly efficient in treatment of herniated cervical discs and enables achieving clinical outcomes associated with much less surgical trauma. The study demonstrates not only the efficacy of the suggested technique but also its safety compared to that of traditional anterior microsurgical techniques that usually involve interbody fusion. This surgery surpasses other interventions in the rate of rehabilitation and social adaptation of patients as well as reduces postoperative hospital stay.

VARIABILITY OF CENTROMERIC HISTONE H3 VARIANTS AND PHYLOGENETIC RELATIONSHIPS IN SECALE.

We examined nucleotide sequence variation in centromeric histone H3 (CENH3) in fifteen rye species and subspecies, including annuals, perennials, self-pollinating and cross-pollinated plants. Levels of genetic variation within N-terminal tail (NTT) and histone fold domain (HFD) were estimated as average per site pairwise nucleotide diversity (p) and as the number of segregating polymorphic sites (S). A comparison of nucleotide diversity (ptot) for NTT and HFD showed that estimates of diversity are consistently greater in NTT. Our estimates of NTT nucleotide diversity for perennial Secale strictum C. Presl were similar to those observed in annual S. cereale L., elevating those estimates for ancient species S. silvestre. A similar pattern of nucleotide polymorthism was observed between the self-pollinating and cross-pollinated subspecies. These results suggest that lower nucleotide diversity within CENH3 for some wild and cultivated subspecies of Secale may be attributable to narrow ecological niche, either evolutionary age or breeding.

CHARACTERIZATION OF CENTROMERIC HISTONE H3 VARIANTS IN ALLOPOLYPLOIDS OF WHEAT AND RYE.

Chromosome elimination of one parental species in hybrid cell observed even after successful fertilization is one of a common phenomenon and the main problems of remote hybridization. Centromeres regulate the process of faithful segregation of chromosomes during cell division. Constant component of the centromeric chromatin is a specialized histone H3 modification (CENH3). CENH3 consists of a conserved C-terminal domain (HFD) and a more variable N-terminal tail (NTT), which plays an important role for CENH3 loading in centromeric chromatin during cell division. In the present study, we performed comparative analysis of CENH3 variants expression in allopolyploids, as well as in different parental varieties of rye (Secale cereale L.) and wheat (Triticum aestivum L.). The varieties of both genera were used as maternal plants. In the coding sequences of the NTT CENH3 we identified the full-length copies of 216 bp with characteristic single nucleotide polymorphisms (SNPs), and copies with 21 bp or 66 bp deletions. The wheat and rye NTT CENH3 copies with 21 bp deletion have not differences in the nucleotide sequences, the rye copies with 66 bp deletion reveal two specific SNPs, which were also found in plant of wheat-rye allopolyploids having 57 chromosomes. The full-length sequences of rye NTT CENH3 show specific SNPs, which were also detected in the full-length sequences of all analyzed plants of wheat-rye allopolyploids. These results indicate that expression of CENH3 copies belonging to each parent was observed in hybrid genomes of aneuploids which arose from octoploid triticale. The NTT CENH3 copies with deletions were not found in secalotriticum (Secalotriticum). The full-length sequences show two rye specific SNPs. Their frequency in secalotriticum significantly exceeds that in hexaploid triticale Mikhas. This fact points to the preferential synthesis of the full NTT CENH3 copies of the rye parental variety in the secalotriticum genome.

[Length and structure of telomeric DNA in three species of Baikal gastropods (Caenogastropoda: Hydrobioidea: Benedictiidae)].

The structure of telomeric repeat (TTAGGG)n was determined and the length of telomeric DNA (tDNA) was measured in three species of gastropods from the family Benedictiidae that are endemic to Lake Baikal. Fluorescence in situ hybridization (FISH) confirmed the localization of a telomeric repeat at the chromosome ends. The sizes of tDNA in "giant" eurybathic, psammo-pelobiontic species Benedictia fragilis and shallow water litho-psammobiontic species B. baicalensis with medium shell sizes were similar (16 ± 2.9 and 15 ± 2.1 kb, respectively), but they had a greater length than that of the shallow water spongio-litobiontic species Kobeltocochlea martensiana with small shells (10.5 ± 1.5 kb). We discuss tendencies in age-related changes in tDNA length in snails and a possible mechanism for maintaining tDNA size in ontogeny.

[A comparative analysis of the effectiveness and potential of endoscopic and microsurgical resection of disc herniations in the lumbosacral spine].

The issue of advantage of endoscopic treatment of spinal disc herniations is debatable. Throughout the development, endoscopic technologies have been compared to microsurgical methods. The two-year experience of applying endoscopic methods was analyzed. The study included 183 patients. The effectiveness of the performed treatment was evaluated according to the MacNab scale of surgical treatment outcomes. Good and excellent results were obtained in 170 cases, which amounted to 92.9%. This cure rate was compared to the similar rate for good and excellent results of the microsurgical treatment method derived from the literature data. The article by American authors who conducted a multicenter study (Lumbar microdiscectomy: a historical perspective and current technical considerations. Koebbe C.J., Maroon J.C., Abla A., El-Kadi H., Bost J. Neurosurg Focus 2002 Aug 15; 13(2): E3) was used. On the basis of this study, the data on higher effectiveness of endoscopic discectomy compared to the microsurgical technique were obtained. The technical capabilities of the endoscopic method for removing spinal disc herniations in comparison to minimally invasive microsurgical techniques were carefully analyzed. It was noted that there were no significant instrumental limitations for using endoscopic techniques, while angled optics and excellent color rendition enable better visualization of the surgical wound structures and more efficient use of the approach space. Given that the technical characteristics and capabilities of this method are not inferior to those of the microsurgical technique, the former technology can be used instead of the standard technique for removing intervertebral disc herniations. Furthermore, the technical capabilities of the method allow performing wide decompression of the neural structures during surgery, which can be used to treat spinal stenoses.

[Organization and evolution of the subtelomeric regions of the rye chromosomes].

Subtelomeric regions of chromosomes are particularly dynamic and variable parts in the evolution of the eukaryotic genomes. A specific feature of the rye (Secale) chromosomes is large heterochromatin blocks at the arms of all seven pairs of chromosomes. Within the genus Secale, an interspecific variation in the genome size reaches nearly 15% between Secale cereale (cultivated rye) and the ancient species S. silvestre and an increase in the size of subtelomeric heterochromatin regions is the main contributor to these differences. Earlier works have demonstrated that the subtelomeric hetechromatin of rye is enriched for a few multicopy tandemly organized DNA families which form the long arrays of monomers. Here we aimed to clarify the fine large-scale organization and mutual arrangement within the tandem arrays of these families and the flanking genomic nonarray DNA. Restriction analysis of the BAC-clones containing genetic material of the short arm of the first rye chromosome (1RS) showed that within arrays the bulk of monomers is organized in the specific higher-order repeat units (up to hexamers) which are generated in the central part of tandem arrays, while only monomers and dimers are present near the boundaries. Sequencing of the genomic nonarray DNA flanking the tandem arrays has demonstrated that this DNA in the studied BAC-clones consists completely of retrotransposons of various classes which are also present in the wheat and barley genomes. Thus, only an explosive amplificcation ofpSc200 and pSc250 monomers, on the background of a saturated mixture of various retrotransposons, can be regarded as a specific molecular process in formation of subtelomeric hetechromatin during the divergence of rye genome from the common ancestor of cereals. Evidently, this process resulted in the enlargement of subtelomeric hetechromatin of S. cereale and an increasing of its genome size.

[Telomere length and phylogenetic relationship of Baikal and Siberian planarians (Turbellaria, Tricladida)].

Dynamics of the telomeric DNA (tDNA) and the phylogeny of the Baikal and Siberian planarians have been studied based on the analysis of the 18S rDNA and beta-actin gene fragments. A relationship between tDNA and the planarians size has been demonstrated. Giant planarians with a minor exception have longer tDNA than little planarians. Phylogenetic affinity between the species that have the stretched tracks of tDNA, big size and similar habitats may indicate possible role of tDNA in the development of the indefinite regenerative capacity of planarians.

[Heterogeneous organization of a tandem repeat family in subtelomeric heterochromatin of rye].

The presence of tandem repeat multicopy families in subtelomeric regions of all chromosomes is a characteristic feature of the rye karyotype, in contrast to the organization of these regions in chromosomes of extensively studied species, such as human, rice, and arabidopsis. To study the molecular structure of these regions, we analyzed BAC clones from a library constructed from the genetic material of rye chromosome 1 short arm (1RS). Screening of the library detected numerous clones that contained copies of multicopy tandem families of DNA sequences pSc200, pSc250, and pSc119.2. An examination of the molecular organization of tandem stretches of the pSc200 family, which is the most common in the rye genome, showed that the subtelomeric 1RS region includes several such stretches, each of which contains characteristic blocks of multimers of various periodicity. Such pattern of heterogeneous organization of tandem repeat stretches differs from the view of the tandem stretches as monotonous sequence of identical monomers, which was generally accepted in recent past.

[Relationship between two distinct families of tandem repeats in rye heterochromatin].

BAC library constructed from the short arm of the first rye (Secale cereale L.) chromosome 1R was screened extensively in order to identify clones containing the copies of a tandemly organized repetitive DNA family, pSc200, which is the most abundant in the rye subtelomeric heterochromatin. Molecular organization of the monomers array and adjacent sequences have been studied in BAC 126/C20. The pSc200 array does not demonstrate the higher-order organization under treatment by different restrictases. The DNA adjacent to the pSc200 array consists of the different repeats included retrotransposon derivatives and another tandemly repeated XbaI family with monomer length of 576 bp, 475 of these show 82% similarity to part of the long terminal repeat of known retrotransposon Cereba. The sequencing of the 13 kb region in BAC 126/C20 revealed the direct junction of the pSc200 and XbaI monomers. The junction between these monomers was abrupt in the identical AT-rich site, CAAAAAT. Another recombinational signal is the palindromes in the close proximity to the site ofjunction. The presence of microhomologies promotes the efficiency action of the proteins involved in the mechanisms of double strand DNA break repair. To our knowledge, for the first time we revealed the direct junction of the monomers, which are longer than 100 bp length and belong to distinct families of tandem repeats from plant genomes.

[A study of dissolved oxygen flux across the water-bottom interface in the northeastern Black Sea (in the region of Dzhubga village)].

Oxygen exchange at the water-bottom interface in the northeastern Black Sea was studied using bottom tanks (fluxes and oxygen consumption for organic matter mineralization and for respiration of soil and water organisms). The relationship of biogenic fluxes and patterns of biochemical (enzyme) destruction of organic matter by the components of the bacterial and microproducer community was established. The prevalence of microbial oxidation of organic matter correlated with high proteolytic activity in near-bottom water. The principal significance of organic matter oxidation in near-bottom water for the phytoplankton and its respiratory expenditures was demonstrated both in the open system of the near-bottom layer and in the closed tank system. A similar trend was demonstrated for benthic organisms.

[Epigenetics of specific chromosome regions].

Investigation of protein complexes, various types of protein modifications and the structure of the chromatin of specific chromosome regions, such as centromeres, telomeres, and adjacent heterochromatic regions, considerably complicated the notion on DNA, prevailing five decades ago, as molecules that exclusively control coding and realization of genetic information. Striking plasticity of the primary structure of centromeric and telomeric DNA suggests a variety of molecular mechanisms underlying fundamental and universal functions of these key chromosome regions. The present review is an attempt to consider the current concepts on the structure of the DNA and protein components, as well as the structure of the chromatin of specific chromosome regions in eukaryotes, and the concerted evolution of these components, leading to the formation of a hierarchy of coordinated DNA-protein complexes.

Different patterns in molecular evolution of the Triticeae.

A huge part of the genomes of most Triticeae species is formed by different families of repetitive DNA sequences. In this paper the phylogenetic distribution of two major classes of the repeats, retrotransposons and tandemly organized DNA sequences, are considered and compared with the evolution of gene-rich regions and generally accepted Triticeae phylogenetic relationships. In Hordeum, LTR-containing retrotransposons are dispersed along the chromosomes and are consistent with the existing picture of the phylogeny of Hordeum. Another retrotransposon class, LINEs, have evolved independently from LTR-retrotransposons. Different retrotransposon classes appear to have competed for genome space during the evolution of Hordeum. Another class of repeats, tandemly organized DNA sequences, tends to cluster at the functionally important regions of chromosomes, centromeres and telomeres. The distribution of a number of tandem DNA families in Triticeae is not congruent with generally accepted phylogenetic relationships. While natural selection is the dominant factor determining the structure of genic regions we suggest that the contribution of random events is important in the evolution of repetitive DNA sequences. The interplay of stochastic processes, molecular drive, and selection determines the structure of chromosomal regions, notably at centromeres and telomeres, stabilizing and differentiating species-specific karyotypes. Thus, the evolution of these regions may occur largely independently of the evolution of gene-rich regions.

Rye chromosome variability in wheat-rye addition and substitution lines.

In a study of polymorphism and stability in rye chromosomes, three rye varieties and the sets of wheat-rye addition and substitution lines were compared using two non-homologous highly repetitive DNA families, pSc200 and pSc250. The rye varieties, Petkus, Imperial and Onohoiskaya, showed polymorphism for the presence and the size of the pSc200 in-situ hybridization signals on chromosome pairs, 2R, 4R and 7R, and the pSc250 signals on chromosomes, 5R, 6R and 7R. Chromosome 1R was heteromorphic within the Onohoiskaya variety. Differences in the distribution of chromosome polymorphisms imply that intervarietal changes to these highly repetitive DNA families occurred independently, despite their juxtaposition or even overlapping locations in subtelomeric heterochromatic regions. In the set of Saratovskaya 29 wheat/Onohoiskaya substitution lines, only chromosome 2R was altered relative to its counterpart in the parental rye variety due to amplification of the pSc250 signal on the long arm, although this did not exceed intervarietal polymorphism. In the set of Chinese Spring wheat/Imperial addition lines, only two Imperial chromosomes, 4R and 6R, were unchanged. We detected the loss of one or both rye homologous chromosomes, the loss of one arm, and the deletion of subtelomeric heterochromatin accompanied by the loss of the pSc200 signal. The results show that Saratovskaya 29/Onohoiskaya chromosome substitution lines possess increased chromosome stability compared with Chinese Spring/Imperial addition lines.

Heterogeneity of the internal structure of PDR1, a family of Ty1/copia-like retrotransposons in pea.

We characterised the extent of heterogeneity among PDR1 elements, a Ty1/copia-like retrotransposon family in pea, by restriction mapping and PCR with primers designed to amplify four functional domains. The data suggest that two main subfamilies of PDR1 differ in the size of their 5'-region. There are also sequence variants and rearranged copies which include a wide range of deletions of different sizes and deletions combined with insertions of host DNA, or inversions of various regions of the retrotransposon. A deletion hotspot has been found at nucleotide position 394, where buffer sequences of 26 bp and 38 bp containing microsatellite motifs have been generated. There is more heterogeneity in the gag domain of PDR1 than in other functional domains, and the extent and pattern of this diversity was assessed among 56 Pisum accessions. We found a higher rate of rearrangement and sequence variation within the gag domain of PDR1 in P. fulvum and P. abyssinicum accessions than would be expected from the degree of insertion site polymorphism. A neighbour-joining phylogenetic tree constructed for gag sequences has a similar branching pattern to the equivalent insertion site tree, implying that the PDR1 family and its gag domain have coevolved with the pea genome. Combining both trees revealed clear and distinct subgroups among the Pisum ssp.

Polymorphism of insertion sites of Ty1-copia class retrotransposons and its use for linkage and diversity analysis in pea.

A sample of 15 cultivars and 56 Pisum accessions from the JIC germplasm core collection has been studied using a modification of the SSAP (sequence-specific amplification polymorphisms) technique; the specific primer was designed to correspond to the polypurine tract (PPT) of PDR1, a Ty1-copia group retrotransposon of pea. Most of these SSAP products were shown to be PDR1 derived. The PDR1 SSAP markers are more informative than previously studied AFLP or RFLP markers and are distributed throughout the genome. Their pattern of variation makes them ideal for integrating genetic maps derived from related crosses. Data sets obtained with AFLP and PDR1 SSAP markers were used to construct neighbour-joining trees and for principal component analysis. These data sets give greater resolution than hitherto available for the characterisation of variation within Pisum, showing that the genus has three main groups: P. fulvum, P. abyssinicum and all other Pisum spp. P. abyssinicum is not a subgroup of cultivated P. sativum, as was previously thought, but has probably been domesticated independently. Modern cultivars are shown to form a single group within Pisum as a whole.

Comparative analysis of the nucleosomal structure of rye, wheat and their relatives.

Analysis of the structure of chromatin in cereal species using micrococcal nuclease (MNase) cleavage showed nucleosomal organization and a ladder with typical nucleosomal spacing of 175-185 bp. Probing with a set of DNA probes localized in the authentic telomeres, subtelomeric regions and bulk chromatin revealed that these chromosomal regions have nucleosomal organization but differ in size of nucleosomes and rate of cleavage between both species and regions. Chromatin from Secale and Dasypyrum cleaved more quickly than that from wheat and barley, perhaps because of their higher content of repetitive sequences with hairpin structures accessible to MNase cleavage. In all species, the telomeric chromatin showed more rapid cleavage kinetics and a shorter nucleosome length (160 bp spacing) than bulk chromatin. Rye telomeric repeat arrays were shortest, ranging from 8 kb to 50 kb while those of wheat ranged from 15 kb up to 175 kb. A gradient of sensitivity to MNase was detected along rye chromosomes. The rye-specific subtelomeric sequences pSc200 and pSc250 have nucleosomes of two lengths, those of the telomeric and of bulk nucleosomes, indicating that the telomeric structure may extended into the chromosomes. More proximal sequences common to rye and wheat, the short tandem-repeat pSc119.2 and rDNA sequence pTa71, showed longer nucleosomal sizes characteristic of bulk chromatin in both species. A strictly defined spacing arrangement (phasing) of nucleosomes was demonstrated along arrays of tandem repeats with different monomer lengths (118, 350 and 550 bp) by combining MNase and restriction enzyme digestion.

[Structural features of the modified BARE-retroelement in the barley (Hordeum vulgare L.) genome]. / Osobennosti struktury modifitsirovannogo BARE-retroélementa genoma iachmenia (Hordeum vulgare L.).

The primary structure of the 4.2-kb BamHI-fragment occurring abundantly in the genome of barley Hordeum vulgare was determined. By means of computer analysis, considerable homology was found between this fragment and the copia-like BARE-l retrotransposon studied earlier. A unique distinction of the BamHI fragment is its symmetrical structure caused by the presence of two mutually inverted parts, each of which is homologous to a long region of BARE-l including a 5'-LTR (long terminal repeat) and the adjacent leader sequence. No sequences homologous to the coding domains of BARE-l were revealed in the fragment. However, potentially functional signals were found: TATA boxes and primer-binding sites (PBS) exhibiting statistically significant homology to the corresponding regulatory signals of known retroelements. Thus, we have revealed and characterized a repeated element of the H. vulgare genome that is a markedly modified derivative of the BARE family of retrotransposons of this genome.