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In this work, we synthesized and characterized the properties of a series of new fluorescent DB3(n) narrow-groove ligands. DB3(n) compounds based on dimeric trisbenzimidazoles have the ability to bind to the AT regions of DNA. The synthesis of DB3(n), whose trisbenzimidazole fragments are linked by oligomethylene linkers of different lengths (n = 1, 5, 9), is based on the condensation of the MB3 monomeric trisbenzimidazole with α,ω-alkyldicarboxylic acids. DB3 (n) proved to be effective inhibitors of the catalytic activity of HIV-1 integrase at submicromolar concentrations (0.20-0.30 μM). DB3(n) was found to inhibit the catalytic activity of DNA topoisomerase I at low micromolar concentrations.

Introduction: A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a weak immune response and require the use of adjuvants. The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid., Materials and Methods: An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated., Results: Mice immunization with rNS5B in a mixture with dnC60 at doses of 250 g/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 720 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60., Conclusions: Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.

The investigation of plant genomes is of great importance for basic research and practical breeding. In 1977, F. Sanger proposed a DNA sequencing method, which allowed the complete sequences of a number of genomes to be determined. Then high-throughput and cost-effective next-generation/second-generation sequencing methods, producing up to billions of short reads, made it possible to sequence genomes of a significant number of species and provided a breakthrough in plant genetic studies. Finally, third-generation sequencing technologies allowed the determination of single-molecule sequences up to a million nucleotides in length, which is key for high-quality genome assemblies. An important task is to obtain a pan-genome, which includes an entire set of nucleotide sequences presented in various genotypes of the same species. The sequencing of plant genomes made it possible to assess intraspecific polymorphism, identify key genes influencing the formation of significant features, and develop molecular markers of economically valuable traits and this has become the basis for the development of marker-assisted and genomic selection. This review provides information on the latest advances in sequencing technologies and the assembly of plant genomes, as well as the opportunities that they open up for basic and applied works.

The objective of the study was to pre-evaluate the applicability of gender-specific nucleotide sequences in human neuroligin genes as alternative DNA markers of sex. A new polymorphic locus based on NLGNX and NLGNY genes was proposed to establish the sex attribute of human biomaterials. The significant difference in the location of these loci relative to the pseudoautosomal region (PAR), as well as the combination of different types of polymorphism on the one hand, and the possibility of using gender-specific primers «in one assay» on the other hand, warrants their use as an additional marker of human sex attribute, including utilization as part of systems for DNA registration in the population. The introduction of a new polymorphic locus based on the NLGNX and NLGNY genes will make it possible to reliably identify the sex attribute of biological material recovered from crime scenes.

The application of array-based oligonucleotides in biological studies is described. These oligonucleotides are mainly used to design large libraries of various nucleotide sequences, which are applied to study protein-nucleic acid interactions, splicing, transcription, translation, and other regulatory processes in mammalian, yeast, and bacterial systems. The application of gene libraries generated by array-based nucleotides along with advanced methods of the combination of DNA duplexes will make it possible to obtain complex genetic designs for synthetic biology.

This paper reports the analysis of the nucleotide sequences of the 5'-untranslated region (5'-UTR) of tick-borne encephalitis virus (TBEV) genomic RNA isolated from 39 individual taiga ticks collected in several regions of Northern Eurasia. The sequences of 5'-UTRs of the Siberian and Far East TBEV genotypes were 89% and 95% identical to the prototype strains (Zausaev and 205), respectively. The detected nucleotide substitutions were typical for these two TBEV genotypes, which made possible unambiguous identification. Both conservative and variable motifs were detected in the 5'-UTR RNA. The B2, C1, and C2 elements of the Y-shaped 5'-UTR structure and the presumable viral RNA-dependent RNA-polymerase binding site were the most variable. The A2, CS A, CS В elements as well as the start codon were conservative. Interestingly, five substitutions in the 5'-UTR C1 variable element of the TBEVs isolated in different geographical regions were strictly conservative, while 11 different substitutions were detected in this element among the laboratory TBEV variants. A little less that a third of all nucleotide substitutions were mapped outside the main elements of the Y-shaped structure. In general, nucleotide substitutions were localized to stem structures, not being found in the hairpin regions of the TBEV 5'-UTR. The results indicated significant variability of the genomic RNA 5'-UTR in the TBEV laboratory strains and field isolates obtained from different geographical regions. It has been suggested that genetic variability of 5'-UTR is characteristic of the TBEV genome 5'-UTR organization and may serve as a structural basis for virus efficient replication in various avian, mammalian, and ixodic tick cells.

De novo assembled transcriptomes of the marine microalga Dunaliella tertiolecta (Chlorophyta) were analyzed. Transcriptome assemblies were performed using short-read RNA-seq data deposited in the SRA database (DNA and RNA Sequence Read Archive, NCBI). A merged transcriptome was assembled using a pooled RNA-seq data set. The goal of the study was in silico identification of nucleotide sequences encoding P-type ATPases in D. tertiolecta transcriptomes. P-type ATPases play a considerable role in the adaptation of an organism to a variable environment, and this problem is particularly significant for microalgae inhabiting an environment with an unstable ionic composition. Particular emphasis was given to searching for a sequence coding Na^(+)-ATPase. This enzyme is expected to function in the plasma membrane of D. tertiolecta like in some marine algae, in particular, in the closely related alga Dunaliella maritima. An ensemble of 12 P-type ATPases consisting of members belonging to the five main subfamilies of the P-type ATPase family was revealed in the assembled transcriptomes. The genes of the following P-type ATPases were found: (1) heavy metal ATPases (subfamily PIB); (2) Ca^(2+)-ATPases of SERCA type (subfamily P2A); (3) H^(+)-ATPases (subfamily P3); (4) phospholipid-transporting ATPases (flippases) (subfamily P4); (5) cation-transporting ATPases of uncertain specificities (subfamily P5). The presence of functional Na^(+)-ATPases in marine algae is presently undoubted. However, contrary to expectations, we failed to find a nucleotide sequence encoding a protein that could unequivocally be considered a Na^(+)-ATPase. Further study is necessary to elucidate the roles of in silico revealed D. tertiolecta ATPases in Na^(+) transport.

A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2-35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region.

Aim: To identify the association between homozygous deletion genotypes of glutathione transferase genes GSTT1 (glutathione transferase theta 1), GSTM1 (glutathione S-transferase mu1) and infertility in Russian men., Materials and Methods: The article presents a comparative analysis of the incidence of homozygous deletion genotypes of glutathione transferase genes GSTM1 and GSTT1 in Russian men with and without infertility. The study group comprised 160 infertile Russian men of reproductive age (mean age 30.2+/-3.6 years.) The infertility diagnosis was verified according to the WHO guidelines. The control group comprised 104 healthy Russian volunteers (mean age 31.3+/-5.4 years.) Molecular genetic detection of GSTM1 and GSTT1 deletion polymorphisms was performed using PCR. The genomic DNA for the study was extracted from whole blood samples., Results: The study and control group differed significantly in incidence of GSTM1 (p=0.043) and GSTT1 (p=0.008) deletion polymorphisms. The probability of detecting "zero" genotypes of the GSTT1 and GSTM1 genes in infertile men was 2.5 (p<0.05) and 1.7 times higher (p<0.05), respectively, than in fertile men., Conclusions: Therefore, the study findings allow us to conclude that the deletion genotypes of GSTM1 and GSTT1 are associated with infertility in Russian men. Molecular genetic analysis of deletion polymorphism of glutathione transferase genes can be recommended for a comprehensive examination of infertile men.

Introns can frequently enhance transgene expression, and sometimes they are absolutely substantial. Based on an analysis of murine genes, in which mRNA does not have alternative splicing, a universal design of the efficiently spliced artificial introns of small sizes has been proposed. These introns are shown to be efficiently spliced in CHO cells from hamster ovaries. The proposed strategy can be used to include introns in cDNA, which would elevate the production of recombinant proteins in cell culture, as well as in transgenic animals.

The discovery of CRISPR/Cas9 brought a hope for having an efficient, reliable, and readily available tool for genome editing. CRISPR/Cas9 is certainly easy to use, while its efficiency and reliability remain the focus of studies. The review describes the general principles of the organization and function of Cas nucleases and a number of important issues to be considered while planning genome editing experiments with CRISPR/Cas9. The issues include evaluation of the efficiency and specificity for Cas9, sgRNA selection, Cas9 variants designed artificially, and use of homologous recombination and nonhomologous end joining in DNA editing.

MuDR exhibits the highest transposition activity and insertional mutagenesis frequency in Mutator (Mu) family. If we isolate the MuDR­insertion­specific flanking sequences (MuDRFs), it will be crucial for using Mu element­mediated mutants. The MuDR­TAIL­PCR system was constructed and optimized using a combination of MuDR­TIR­nested specific primers and 12 arbitrary degenerate (AD) primers, modified reaction system and procedure and mutant DNA templates of 87 genotypes from M2 or M2:3 families created by crossing the W22::Mu line (active MuDR donor parent) from the UniformMu population with the Zong31 (Z31) line (recipient parent). Here 129 different MuDRFs were acquired by MuDR­TAIL­PCR, accounting for 86.60 % of the total mutant­specific agarose gel bands. In addition, we confirmed the authenticity of the non­redundant flanking sequence amplifications. The amplified non­redundant flanking sequences accounted for 65.12 % of the total MuDRFs, and 88.00 % of the non­redundant MuDRFs were inserted inside the genes. These results show that the MuDR­TAIL­PCR system that we developed can be used for specifically isolating MuDRFs.

A novel AluI-polymorphism in the fourth intron of chicken growth hormone gene was shown. It was detected the cytosine to thymine transition in the restriction site for AluI. Primers, that flanking the 460 bp fragment of the fourth intron, containing a polymorphic restriction site for AluI, was designed. The nucleotide sequence fragments amplified polymorphic variants was determined. Using designed primers was analyzed the genetic structure of populations of White Plymouth Rock, Poltava Clay, Rhode Island Red and Borkovskaya Barvistaya chicken breeds. It was found that growth hormone gene (by AluI-polymorphism in the fourthintron) was polymorphic in all experimental populations. Frequencies of alleles C and T in chicken population of White Plymouth Rock breed were 0,14 and 0,86; Rhode Island Red – 0,3 and 0,7; Poltava Clay – 0,04 and 0,96; Borkovskaya Barvistaya – 0,08 and 0,92 respectively. The tendency to increase egg production and egg weight of chicken with C/C genotype, as well as meat quality (live weight, carcass weight, weight of pectoral muscles) of chickens with genotype T/T of Rhode Island Red chicken breed was shown.

Development of RNAi-based therapeutics is a fast growing field of pharmaceutical industry. Using plants for production of pharmaceutically valuable siRNAs may have significant advantages of cost-effectiveness, scalability and low risk of contamination with human pathogens. If edible plant species are genetically engineered to synthesize siRNAs, the costly stage of target product purification may be omitted. We describe the establishment of transgenic lettuce plants producing shRNA targeting delta isoform of protein kinase C (PKC-delta), an effective target for RNAi-based treatment of arterial hypertension. Transgenic lettuce plants were obtained by Agrobacterium-mediated transformation with genetic constructs harboring antiPKC and scrambled (control) shRNA genes. The presence of transgenes was proved by PCR analysis, and the accumulation of antiPKC shRNA was estimated using RT-qPCR technique. Six transgenic lettuce lines showed varying levels of antiPKC shRNA expression with the highest value reaching 14 ± 9 % of highly abundant endogenous lettuce micro RNA (miR156a), or 12.7 fmol/g dry weight. Plants carrying either antiPKC or scrambled shRNA genes flowered normally, but did not produce seeds. The described transgenic lettuce plants accumulating antiPKC siRNA are the subject for animal testing and can be considered as a raw material for the development of novel antihypertensive drugs.

Testicular cancer is the most common form of solid cancer in young men. Testicular cancer is represented by testicular germ cell tumors (TGCTs) derived from embryonic stem cells with different degrees of differentiation in about 95% of cases. The development of these tumors is related to the formation of a pool of male germ cells and gametogenesis. Clinical factors that are predisposed to the development of germ-cell tumors include cryptorchidism and testicular microlithiasis, as well as infertility associated with the gr/gr deletion within the AZFс locus. KITLG, SPRY4, and BAK1 genes affect the development of the testes and gametogenesis; mutations and polymorphisms of these genes lead to a significant increase in the risk of the TGCT development. To determine the relationship between gene polymorphisms and the development of TGCTs, we developed a system for detection and studied the allele and genotype frequencies of the KITLG (rs995030, rs1508595), SPRY4 (rs4624820, rs6897876), and BAK1 (rs210138) genes in fertile men, patients with TGCTs, and patients with infertility that have the AZFс deletion. A significant association of rs995030 of the KITLG gene with the development of TGCTs (p = 0.029 for the allele G, p = 0.0124 for the genotype GG) was revealed. Significant differences in the frequencies of the studied polymorphisms in patients with the AZFc deletion and the control group of fertile men were not found. We showed significant differences in the frequencies for the combination of all high-risk polymorphisms in the control group, patients with the AZFc deletion and patients with TGCTs (p (TGCTs-AZF-control) = 0.0207). A fivefold increase in the frequency of the combination of all genotypes in the TGCT group (p = 0.0116; OR = 5.25 [1.44-19.15]) and 3.7-fold increase was identified in patients with the AZFc deletion (p = 0.045; OR = 3.69 [1.11-12.29]) were revealed. The genotyping of patients with infertility caused by the AZFc deletion can be used to identify individuals with an increased risk of TGCTs.

In the previous cytogenetic study of new human stem cell line 4BL at the 205th passage we observed the ploidy of chromosomal set and regular aberrations. To investigate the nature of monosomy of certain chromosomes the array CGH and FISH analyses have been used. The aberrations of chromosomes have been identified in all the cases of monosomies previously revealed by G-banding. The largest changes of the DNA balance have been detected in the chromosomes 2, 4, 10, 13 and 17. The probable cause of the monosomies of chromosomes 4, 10, 13 and 17 is massive loss of the genetic material. The monosomy of the second chromosome pair is caused by significant transformation one of the homologs in a type of numerous duplications and formation of der(2)t(2;?)(q21;?). Due to application of array CGH the regions of the structural aberrations of the chromosomes 2, 4, 10, 13 and 17 have been concretized, what permitted to perform their clarifying identification by multicolored FISH method. The results obtained by us confirm the hypothesis about coordinated appearance of the deletions and duplications and their stabilization impact on the transformed chromosomes.

The identification of probiotic strain Lactobacillus sp. 55 at species level on the basis of analysis of the 16S rRNA gene nucleotide sequences has been done. It has been established that investigated strain belongs to the species Lactobacillus gasseri. The nucleotide sequence of strain Lactobacillus gasseri 55 16S rRNA gene’s fragment has been deposited in international database GenBank (NCBI) under number 314159 KT.

The analysis of Ukrainian isolate of beet necrotic yellow vein virus has been performed. The partial nucleotide sequence of cDNA corresponding to RNA-2 of BNYVV isolates were analyzed and Ukrainian isolate AG9 of BNYVV was assigned to type A strains based on DNA sequences. The nucleotide sequence of gene encoding a coat protein of Ukrainian isolate of BNYVV was compared with appropriate nucleotide sequences existing in the GeneBank and the phylogenetic analysis of investigated virus was done. It was shown that Ukrainian isolate AG9 of BNYVV has 100 % homology to isolate originating from Sweden.

Aim: Confirmation of taxonomic position of Lactobacillus fermentum 90 TC-4 strain using phenotypic (classic microbiological, MALDI TOF mass-spectrometry) and genetic (16S rRNA gene segment sequencing and full genome sequencing) methods., Materials and Methods: Object of the study--Lactobacillus fermentum 90 TC-4 strains from various collections. Mass-spectrometric analysis was carried out using Autoflex MALDI TOF mass-spectrometer (Bruker Daltonics, Germany), study of biochemical properties of the strain was carried out using API 50 CHL strips (Biomerueux, France), "DNA-sorb B" kitwas used for isolation ofgenome DNA (CRIE, Moscow). Sequencing of the accumulated fragments of 16S rRNA gene was carried out using GenomeLab GeXP sequencing (Beckman Coulter, USA), full genome sequencing was carried out in MiSeq platform (Illumina). Assembly of genome and bioinformation analysis was carried out using BLAST program (www.blast.ncbi.nlm.nih.gov/blast.cgi), "CLC Bio Assembly" and genome server RAST (rast.nmpdr.org)., Results: L. fermentum 90 TC-4 strain was established to be contaminated by L. plantarum culture in a series of cases. As a result of identification of a pure culture of L. fermentum 90 TC-4 strain using a specter of high-technology methods, membership of the strain in L. fer- mentum species has been proven., Conclusion: Taxonomic status of L. fermentum 90 TC-4 strain was confirmed.

Molecular genetic techniques and approaches in epidemiological studies were breakthrough in the understanding of the laws, ways, and mechanisms of the spread of the pathogens. However, lack of standard methods makes it difficult to compare results obtained by different scientific groups. In this work we propose to choose one fragment of the TBEV genome as a genetic marker whose sequencing would be both obligatory and sufficient for the molecular epidemiological studies. The best candidate for this purpose may be a fragment of the gene E of 454 nucleotides in length. The deduced amino acid sequence of this fragment was a basis for a new approach for the TBEV differentiation with clusteron being a structural unit (Kovalev and Mukhacheva, 2013). The clusteron approach was proved to be informative for studying the genetic structure of the TBEV-Sib population in the Middle Urals. TBE foci were shown to be unique in both quantitative and qualitative composition of the clusterons. The greatest clusteron diversity in the south of the Middle Urals, through the Trans-Siberian way, may reflect the history of the colonization, closely associated with the roads between Siberia and the European part of Russia. The age of three clusterons did not exceed 50 years, which may indicate an ongoing evolutionary process taking place in the TBEV-Sib populations. In turn, their spatial distribution indicates the crucial role of human factors in the spread of the TBEV (Kovalev & Mukhacheva, 2014). The clusteron approach provides formalization of ideas about the structure of the viral populations and could be used not only by researchers but also by epidemiological surveillance services. Unification of the studies of the TBEV on the basis of a standard genetic marker would consolidate the efforts of researchers from different regions of Russia and other countries.

The MspI-polymorphism in the fourth intron of the growth hormone gene in populations of White Plymouth Rock, Poltava Clay, Rhode Island Red and Borkovskaya Barvistaya chicken breeds was studied. It is shown that in all examined chicken populations the growth hormone gene is polymorphic. It was found that the presence of "additional" phenotype (restriction pattern) is not associated with duplication of the growth hormone gene. The possibility of formation of heteroduplex DNA of two different types in the course of amplification of heterozygous samples B/C, containing the site 'CCGG', which leads to formation the additional DNA fragment which do not contain the site 'CCGG', was described. The nucleotide sequences of alleles A, B, C and "additional" fragment is described. Frequencies of alleles A, B and C in chicken population of White Plymouth Rock breed were 0.56; 0.16 and 0.28; Poltava Clay--0.10; 0.07 and 0.83; Rhode Island Red--0.27; 0.31 and 0.42; Borkovskaya Barvistaya--0.75; 0.08 and 0.17 respectively.

The Acetobacteraceae family of the class Alpha Proteobacteria is comprised of high sugar and acid tolerant bacteria. The Acetic Acid Bacteria are the economically most significant group of this family because of its association with food products like vinegar, wine etc. Acetobacteraceae are often hard to culture in laboratory conditions and they also maintain very low abundances in their natural habitats. Thus identification of the organisms in such environments is greatly dependent on modern tools of molecular biology which require a thorough knowledge of specific conserved gene sequences that may act as primers and or probes. Moreover unconserved domains in genes also become markers for differentiating closely related genera. In bacteria, the 16S rRNA gene is an ideal candidate for such conserved and variable domains. In order to study the conserved and variable domains of the 16S rRNA gene of Acetic Acid Bacteria and the Acetobacteraceae family, sequences from publicly available databases were aligned and compared. Near complete sequences of the gene were also obtained from Kombucha tea biofilm, a known Acetobacteraceae family habitat, in order to corroborate the domains obtained from the alignment studies. The study indicated that the degree of conservation in the gene is significantly higher among the Acetic Acid Bacteria than the whole Acetobacteraceae family. Moreover it was also observed that the previously described hypervariable regions V1, V3, V5, V6 and V7 were more or less conserved in the family and the spans of the variable regions are quite distinct as well.

In the genome of most vertebrates growth-hormone gene is presented in a single copy, while in salmonids after one of the duplication events many genes were multiplied, including growth hormone gene. In salmonids, the growth-hormone gene exists as two independently inherited functional paralogues, gh1 and gh2. In this study, we performed a comparative analysis of gh1 and gh2 growth-hormone genes and their adjacent sequences in Levanidov's charr Salvelinus levanidovi to determine their functionality and define the potential differences. We found that both genes have the same gene structure and are composed of six exons (I-VI) and five introns (A, B, C, D, E). However, the respective gene sequences differ in length. A comparison of exons showed that the size of each exon is identical in both paralogues. The overall length of genes differs due to the varying lengths of introns. Coding sequence of both genes contains an open reading frame for 210 amino acids. We identified regulatory elements in the promoter region of both genes: TATA box, A/T-rich regions that contain binding sites for pituitary-specific transcriptional activator Pit-1, and regions responsible for interaction with other transcriptional activators and initiators, in particular hormone receptors. The obtained data indicate that both genes are functional.

Nucleotide sequences were identified in the eye tissues (lens, retina, and retinal pigment epithelium) of the adult newt Pleurodeles waltl by the polymerase chain reaction with primers for the Ns gene. Sequencing showed that these nucleotide sequences belong to the Ns gene of the newt P. walt, which encodes the nucleolar protein nucleostemin. Structural analysis revealed a high homology of Ns nucleotide sequences of P. walt! with those of newts. Cynops pyrrhogaster and Notophthalmus viridescens. The expression of the Ns gene of P. walt, identified in the specialized eye cells of adult newts of the studied species, indicates that these differentiated cells retain some of the molecular characteristics inherent to the undifferentiated cells.

Ants are one of the most ancient and successful groups of eusocial animals and they are spread all over the world. The nucleotide sequences of the genomes of eight ant species were determined by the year 2014. In these species, the mechanisms of ecological success, cast differentiation, and social communication were studied at genomic level. In ants, the genes of the cytochromes P450 involved in metabolism of xenobiotics and various endogenic substances are amplified. Although the substrates for several cytochrome P450 families have been identified, the functions of the ninth family, which is one of the most amplified, remain unknown. The black garden ant Lasius niger is one of the spices that have successfully adapted to urban conditions. To study the mechanisms of adaptation, we have read and annotated the nucleotide sequence of the L. niger genome; we have predicted the functions of the CYP9 genes using virtual screening. The obtained data allow us to suggest that cytochromes P450 are involved in the metabolism of various xenobiotics such as phytotoxins, mycotoxins, and insecticides. We assume that the functional divergence of the new CYP9 duplications was initially aimed at developing resistance to various mycotoxins, in particular to those produced by Fusarium fungi and, subsequently, to other xenobiotics.

Bacteriophage λ is a classical model object in molecular biology, but little is still known on the physical properties of its DNA and regulatory elements. A study was made of the electrostatic properties of phage λ DNA and regulatory elements. A global electrostatic potential distribution along the phage genome was found to be nonuniform with main regulatory elements being located in a limited region with a high potential. The RNA polymerase binding frequency on the linearized phage chromosome directly correlates with its local potential. Strong promoters of the phage and its host Escherichia coli have distinct electrostatic upstream elements, which differ in nucleotide sequence. Attachment and recombination sites of phage λ and its host have a higher potential, which possibly facilitates their recognition by integrase. Phage λ and host Rho-independent terminators have a symmetrical M-shaped potential profile, which only slightly depends on the annotated terminator palindrome length, and occur in a region with a substantially higher potential, which may cause polymerase retention, facilitating the formation of a terminator hairpin in RNA. It was concluded that virtually all elements of phage λ genome have potential distribution specifics, which are related to their structural properties and may play a role in their biological function. The global potential distribution along the phage genome reflects the architecture of the regulation of its transcription and integration in the host genome.

A study of the ITS1 nucleotide sequences of 1000 moss species of 62 families, 11 liverwort species from five orders, and one hornwort Anthoceros agrestis identified five highly conserved motifs (CM1-CM5), which are presumably involved in pre-rRNA processing. Although the ITS1 sequences substantially differ in length and the extent of divergence, the conserved motifs are found in all of them. ITS1 secondary structures were constructed for 76 mosses, and main regularities at conserved motif positioning were observed. The positions of processing sites in the ITS1 secondary structure of the yeast Saccharomyces cerevisiae were found to be similar to the positions of the conserved motifs in the ITS1 secondary structures of mosses and liverworts. In addition, a potential hairpin formation in the putative secondary structure of a pre-rRNA fragment was considered for the region between ITS1 CM4-CM5 and a highly conserved region between hairpins 49 and 50 (H49 and H50) of the 18S rRNA.

Aim: Demonstrate the possibility of using nested PCR method for determination of Varicella Zoster virus (VZV) in clinical samples of peripheral blood of patients., Materials and Methods: Material from 35 patients with clinical manifestations of herpes zoster and control group of 20 healthy donors was used in the study. Monocyte fraction of venous blood cells, pretreated with heparin, was isolated by centrifugation in ficoll-verografin density gradient, total DNA was then isolated from cells by phenol-chloroform extraction with subsequent precipitation with alcohol. Polymerase chain reaction was carried out in thermocyclers Tercyc and TProfessional Gradient (Biometra), amplified DNA was analyzed by electrophoresis on 1.6% agarose gel in the presence of ethidium bromide., Results: Data on detection of viral DNA in blood monocytes in 17 (49%) of ill patients, as well as in 1 (out of 20 in control group) practically healthy donor were obtained. A possibility of a subclinical reactivation of the virus is discussed in the latter case., Conclusion: A possibility of viral DNA determination in monocytes of patient blood without using expensive equipment is shown, that could find application in clinical practice, especially for diagnostics of patients with non-characteristic clinical manifestations, as well as patients with subclinical forms of the disease.

The complete mitogenome of the peacock sole Pardachirus pavoninus (Lacepède, 1802) was determined. The total length is 16 536 bp, containing 13 protein-coding genes, 22 tRNA genes and two rRNA genes, as well as one control region (CR). The L-strand replication origin (OL), which is typically located in the WANCY cluster, is lost in P. pavoninus. The gene arrangement is identical to that in most teleosts. Comparison of the CR sequences among 13 soles reveals that a 211-bp fragment at the 5'-end of the CR is lost in the P. pavoninus mitogenome, responsible for its short sequence with a length of 872 bp. All typical conservative blocks (TAS, CSB-F, E, D, C, B, A, CSB-1, 2, 3) are identified. Seven out of 13 soles contain tandem repeats in the CR and the possible mechanisms of their formation are discussed. These results may provide the consensus sequences of the conserved units in the sole CR as well as molecular data for phylogenetic studies on Soleidae and Pleuronectiformes.

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol%, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002(T), respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classiffication of strain 32d as a D. maris strain and of strain 263, as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

5S rDNA genes of the stingray Potamotrygon motoro were PCR replicated, purified, cloned and sequenced. Two distinct classes of segments of different sizes were obtained. The smallest, with 342 bp units, was classified as class I, and the largest, with 1900 bp units, was designated as class II. Alignment with the consensus sequences for both classes showed changes in a few bases in the 5S rDNA genes. TATA-like sequences were detected in the nontranscribed spacer (NTS) regions of class I and a microsatellite (GCT) 10 sequence was detected in the NTS region of class II. The results obtained can help to understand the molecular organization of ribosomal genes and the mechanism of gene dispersion.

In the bacterium Azospirillum brasilense Sp245, extracellular calcofluor-binding polysaccharides (Cal+ phenotype) and two types of lipopolysaccharides, LPSI and LPSII, were previously identified. These lipopolysaccharides share the same repeating O-polysaccharide unit but have different antigenic structures and different charges of their O-polysaccharides and/or core oligosaccharides. Several dozens of predicted genes involved in the biosynthesis of polysaccharides have been localized in the AZOBR&#95;p6 plasmid of strain Sp245 (GenBank accession no. HE577333). In the present work, it was demonstrated that an artificial transposon Omegon-Km had inserted into the central region of the AZOBR&#95;p60120 gene in the A. brasilense Sp245 LPSI- Cal- KM252 mutant. In A. brasilense strain Sp245, this plasmid gene encodes a putative glycosyltransferase containing conserved domains characteristic of the enzymes participating in the synthesis of O-polysaccharides and capsular polysaccharides (accession no. YP004987664). In mutant KM252, a respective predicted protein is expected to be completely inactivated. As a result of the analysis of the EcoRI fragment of the AZOBR&#95;p6 plasmid, encompassing the AZOBR&#95;p60120 gene and a number of other loci, novel data on the structure of AZOBR&#95;p6 were obtained: an approximately 5-kb gap (GenBank accession no. KM189439) was closed in the nucleotide sequence of this plasmid.

The microsporidium Glugea gasterostei from the three-spined stickleback Gasterosteus aculeatus was described as an independent species basing upon morphological and ecological traits of the parasite (Voronin, 1974), further supported by ultrastructural characters of its spores (Voronin, 1983). During the revision of microsporidia of the genus Glugea (Canning, Lom, 1986; Lom, 2002), the validity of this species was doubted and it was synonymized with G. anomala. Nevertheless, the molecular phylogenetic analysis performed in the present study showed the unique molecular haplotype of small subunit rRNA gene of G. gasterostei (Genbank accession number KM977990) and its close relatedness to G. anomala, G. atherinae and G. hertwigi (sequence similarity of 99.7 %). One of typical characters of G. gasterostei, as opposed to G. anomala, is the formation of xenomas on inner tissues and not on the surface of infected fishes. This feature is retained even after the infection of different host species. Taken together, these data confirm the validity of G. gasterostei as a separate species among closely related taxa that had diverged comparatively recently.

We performed phylogenetic analysis for ten Pleurotus species, based on internal transcribed spacer (ITS) sequences of rDNA. A phylogenetic tree was constructed on the basis of 31 oyster fungi strains of different origin and 10 reference sequences from GenBank. Our analysis demonstrates that the tested Pleurotus species are of monophyletic origin. We evaluated the evolutionary distances between these species. Classic genetic analysis of sexual compatibility based on monocaryon (mon)-mon crosses showed no reproductive barriers within the P. cornucopiae-P. euosmus species complex. Thus, despite the divergence (subclustering) between commercial strains and natural isolates of P. ostreatus revealed by phylogenetic analysis, there is no reproductive isolation between these groups. A common allele of the matB locus was identified for the commercial strains Sommer and L/4, supporting the common origin of these strains.

Agrobacterium is a genus of soil bacteria with the ability to transform plant cells by a T-DNA-sequence located on the pTi/pRi- plasmid containing a set of genes expressed in plant cells. Expression of these genes leads to a proliferation of transformed cells, with the subsequent formation of tumors or growths of roots and the synthesis of opines--products of the condensation of amino acids with ketoacids or sugars used by Agrobacteria as a source of carbon and nitrogen. In this review, we systematized the information about most common opines in plant--Agrobacterium systems and their biosynthesis and catabolism genes, as well as the role of opines in the interaction of pathogenic Agrobacterium with plants and with other Agrobacterium strains, including the genetic consequences of such interactions.

This work describes the results, of the in silico analysis of the genetic diversity of the citrullinureidase gene (ctu) in two species of bacteria of the genus Francisella: tularensis (ssp. tularensis, holarctica, mediasiatica, novicida) and philomiragia. The strains of the Central Asiatic subspecies possessing the citrullinureidase activity differ in the gene ctu from the ssp tularensis Schu by three nucleotide substitutions leading to two insignificant amino acid substitutions in the encoded polypeptide. In the strain F. tularensis of the ssp. holarctica the gene ctu encodes inactive enzyme, which is probably due to amino acid substitutions: 151 Gly --> Asp, 183 Pro --> Leu, 222 Asp --> Asn. Except for the Japan biovar bacteria, in all strains of the Holarctic subspecies there are two stop codons in the gene ctu. The bacteria of the subspecies novicida contain the ctu gene only in the strain 3523, whereas the other strains contain the gene FTN&#95;0827 encoding the C-N hydrolase, which probably provides the citrullinureidase activity.

Tandemly-repeated sequences represent a unique class of eukaryotic DNA. Their content in the genome of higher eukaryotes mounts to tens of percents. However, the evolution of this class of sequences is poorly-studied. In our paper, 62 families of Mus musculus tandem repeats are analyzed by bioinformatic methods, and 7 of them are analyzed by fluorescence in situ hybridization. It is shown that the same tandem repeat sets co-occure only in closely related species of mice. But even in such species we observe differences in localization on the chromosomes and the number of individual tandem repeats. With increasing evolutionary distance only some of the tandem repeat families remain common for different species. It is shown, that the use of a combination of bioinformatics and molecular biology techniques is very perspective for further studies of the evolution of tandem repeats.

Early stages of human embryonic development are characterized by spatio-temporal coincidence of events of total epigenetic genome reprogramming and elevated level of mosaic forms of numerical chromosome abnormalities. It is possible that the abnormal reprogramming of various regions of the genome can lead to violations of local epigenetic chromatin organization and gene expression, affecting the correct chromosome segregation during mitosis. In this study, a comparative analysis of the methylation index of LINE-1 retrotransposon, which is largely reflecting the methylation profile of the genome, is performed in placental tissues of spontaneous abortions with complete and mosaic forms of aneuploidy, and with a normal karyotype, as well as in the control group of induced abortions of the first trimester of pregnancy. It was shown that extraembryonic mesoderm and chorionic cytotrophoblast of spontaneous abortions with chromosomal mosaicism are characterized by the highest index of LINE-1 methylation among all groups studied. At the same time excessive hypomethylation of transposable genetic element recorded in spontaneous abortions with normal karyotype. It is suggested that violations of parental genomes demethylation during epigenetic reprogramming at preimplantation stages of development may be associated with an increased frequency of mitotic errors in chromosome segregation, leading to the formation of a mosaic karyotype.

The class of tandemly repeated sequences exists only in eukaryotic genomes and absent in prokaryotes. The tens percent of eukaryotic genome are built up of the tandem repeats. The whole set of different tandem repeats is not revealed to any of the eukaryote species in spite of the half century history of its investigation by molecular biology methods. Previously we found the set of tandem repeats in the database of well assembled mouse genome with the bioinformatics methods. In the current work we applied the same methods to the poorly assembled hamster Mesocricetus auratus genome. 19 tandem repeats families have been found in hamster genome by bioinformatics (in silico). Only one of tandem repeats' families found have been cloned previously and exists in the Repbase, the database of all known repetitive fragments. The rest of the families are new and need the experimental verification by FISH (in situ). Oligo probes were designed at the base of in silico found sequences. Oligo probe for the known tandem repeat gives the same signal as the cloned probe, i.e., probes designed are suitable for oligo-FISH. All four oligo probes tested give signal at the heterochromatic centromeric region as expected, though with different intensities and at different number of chromosomes. The results show the power of the in silico methods for the mostly mysterious genome component, tandem repeats, investigation.

The article considers molecular genetic characteristic of RNA of human enterovirus detected in bio-test from child with serous meningitis. The nucleotide sequence of genome DNA is analyzed. In 98% it is identical to corresponding nucleotide sequences of strains of human enterovirus A serotype 71 detected in China.

Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5'-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.

The first data on nucleotide sequences of cytochrome C oxidase I (CO1) gene of Arctic and striped flounders of the Sea of Okhotsk are analyzed. Nucleotide diversity of CO1 gene for 10 individuals of arctic flounder (π = 0,00370) is slightly less than that for 28 individuals of a striped flounder (π = 0,00446), caught in the Taui Lip, the Sea of Okhotsk. The size of the disparity index found on variability of nucleotides at individuals of a polar flounder indicates not selective nature of replacements, whereas at individuals of a striped flounder about a third of replacements is selective. The size of nucleotide distinctions between individuals arctic and striped flounders confirms the specific status of these fishes.

A new aliphatic amidase gene (ami), having a level of similarity with the nearest homologs of no more than 77%, was identified in the Rhodococcus erythropolis TA37 strain, which is able to hydrolyze a wide range of amides. The amidase gene was cloned within a 3.7 kb chromosomal locus, which also contains putative acetyl-CoA ligase and ABC-type transportergenes. The structure of this locus in the R. erythropolis TA37 strain differs from the structure of loci in other Rhodococcus strains. The amidase gene is expressed in Escherichia coli cells. It was demonstrated that amidase (generated in the recombinant strain) efficiently hydrolyzes acetamide (aliphatic anmide) and does not use 4'-nitroacetanilide (N-substituted amide) as a substrate. Insertional inactivation of the amidase gene in the R. erythropolis TA37 strain results in a considerable decrease (by at least 6-7 times) in basal amidase activity, indicating functional amidase activity in the R. erythropolis TA37 strain.

The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

The zona pellucida 3 (ZP3) plays a crucial role in reproductive immunology. We obtained a full-length cDNA encoding Chinese Zokor zp3, using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1269 nucleotides encoding a polypeptide of 422 amino acid residues. The amino acid sequence has a high degree of homology with hamster (78%), mouse (76%), and rat (74%). XhoI and SacI sites restricted 1158 bp fragment of zokor ZP3 cDNA, excluding the signal sequence and transmembrane-like domain was cloned under the phage T7 promoterlac operator control in the pET-28a(+) vector. Recombinant pET-zokorZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strain BL21 (DE3). Optimum expression of r-ZP3 was observed at 28 degrees C, 1 mM IPTG and 2 h of inducing. The purified protein was tested by Western blot.

Data regarding the structure and variation of the nucleotide sequence of the cytochrome b gene of mitochondrial DNA of the Siberian Sucker from the Kolyma River were obtained. Analysis of the median network revealed that evolutionary lines diverged from a common ancestor. Penetration of the sucker into Asia from Northern America took place between the Early and Middle Pleistocene. Prolonged reproductive isolation of the Siberian and Northern American suckers led to interspecies divergence with the appearance of amino acid substitutions, which, apparently, fixed due to positive selection. The Siberian Sucker appeared to have three modifications of the Cytb protein.

Flavobacterium columnare is an important bacterial pathogen of fish with wide ge- netic variability within species. This intraspecies diversity has been termed as genomovars and genomovar groups on the basis of Restriction Fragment Length Polymorphisms of 16S rDNA and 16S-23S rDNA Intergenic Spacer Region (ISR), respectively. In this study, we demonstrate the source of genetic heterogeneity in the F. columnare by sequence analysis of ISR. Length of ISR sequences of different genomovars varied from 553 to 592 nucleotides, while the similarity among sequences ranged from 76.1 to 92.6%. A common ISR structure with tRNAAa and tRNAne embedded within the sequence was identified in all the genomovars ofF. columnare. The results show that strains of F. columnare can be categorized into five genomovar groups based on the heterogeneity in the ISR sequences. Of these, strains belonging to Genomovar I and II can be sub-divided into two groups each; while strains of Genomovar III belonged to one group. Sequence similarity between genomovar groups was lower for ISR (76.1-92.6%) as compared to 16S rDNA (96.1-99.4%) indicating its ability to resolve closely related groups within the genomovars of F. columnare. The main source of variation between genomovar groups is the presence of three hyper variable regions (V1, V2 & V3) in the ISR Of the three, V3 was found to be the most heterogeneous region and was found to be useful in assigning genomovar group to an individual strain of F. columnare.

A complex comparative genetic approach was used for the investigation of the structural and functional diversity of genes for the restoration of sunflower pollen fertility. It includes (i) hybridological analysis; (ii) analysis of polymorphism among EST fragments.homologous to the known Rf genes that contain repeated motives of 35 amino acids (RFL-PPR); (iii) the development of molecular markers. Monogenic segregation in three interline cross combinations and the results of molecular marker analysis confirmed the allelic differences of parental lines in the Mendelian locus for CMS PET1 pollen fertility restoration. Introns were found in two RFL-PPR fragments. Two allelic variants of the QHL12D20 fragment were detected among the sixty lines of the sunflower genetic collection. An intron of QHL12D20 fragment was homologous to an intron of the AHBP-1B gene; the product of this gene-has a similarity with the transcription factor of the bZIP-family of Arabidopsis. A relationship between the QHL12D20 polymorphism and the functional state of the Rfl locus was revealed.

An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.