3,145 results on '"Base sequence"'
Search Results
2. [Adjuvant effect of dispersed fullerene C60 on the immune response to constructs harboring amino acid and nucleotide sequences of hepatitis C virus nonstructural NS5B protein].
- Author
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Masalova OV, Lesnova EI, Andreev SM, Shershakova NN, Kozlov VV, Permyakova KY, Demidova NA, Valuev-Elliston VT, Turetskiy EA, Ivanov AV, Nikolaeva TN, Khaitov MR, Pronin AV, and Kushch AA
- Subjects
- Mice, Animals, Hepacivirus, Base Sequence, Amino Acids genetics, Amino Acids metabolism, Amino Acids pharmacology, Mice, Inbred C57BL, Adjuvants, Immunologic genetics, Immunity, Cellular, Recombinant Proteins genetics, Mice, Inbred BALB C, Fullerenes pharmacology, Fullerenes metabolism, Hepatitis C, Vaccines, DNA genetics, Vaccines, DNA pharmacology, Viral Hepatitis Vaccines genetics, Viral Hepatitis Vaccines pharmacology
- Abstract
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.
- Published
- 2023
- Full Text
- View/download PDF
3. [Plant Genome Sequencing: Modern Technologies and Novel Opportunities for Breeding].
- Author
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Dmitriev AA, Pushkova EN, and Melnikova NV
- Subjects
- Base Sequence, Chromosome Mapping, Plants genetics, Sequence Analysis, DNA methods, Genome, Plant, Plant Breeding
- Abstract
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.
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- 2022
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4. [New polymorphic DNA marker to determine a person's sex from biological material].
- Author
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Garafutdinov RR, Sakhabutdinova AR, Aminev FG, and Chemeris AV
- Subjects
- Base Sequence, Genetic Markers genetics, Humans, DNA genetics, Polymorphism, Genetic
- Abstract
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.
- Published
- 2022
- Full Text
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5. [Application of Array-Based Oligonucleotides for Synthesis of Genetic Designs].
- Author
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Sinyakov AN, Ryabinin VA, and Kostina EV
- Subjects
- Animals, Base Sequence, Gene Library, DNA, Oligonucleotides genetics
- Abstract
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.
- Published
- 2021
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6. [Genetic Variability of Tick-Borne Encephalitis Virus Genome 5'-UTR from Northern Eurasia].
- Author
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Ponomareva EP, Ternovoi VA, Mikryukova TP, Protopopova EV, Tupota NL, and Loktev VB
- Subjects
- Animals, Base Sequence, Genome, Viral genetics, Phylogeny, RNA, Viral genetics, Encephalitis Viruses, Tick-Borne genetics, Encephalitis, Tick-Borne genetics, Ixodes
- Abstract
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.
- Published
- 2021
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7. [In silico Analyses of Transcriptomes of the Marine Green Microalga Dunaliella tertiolecta: Identification of Sequences Encoding P-type ATPases].
- Author
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Popova LG, Belyaev DV, Shuvalov AV, Yurchenko AA, Matalin DA, Khramov DE, Orlova YV, and Balnokin YV
- Subjects
- Adenosine Triphosphatases classification, Adenosine Triphosphatases isolation & purification, Base Sequence, Computer Simulation, Molecular Sequence Annotation, P-type ATPases isolation & purification, Adenosine Triphosphatases genetics, Microalgae genetics, P-type ATPases genetics, Transcriptome genetics
- Abstract
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.
- Published
- 2018
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8. [Bacteriophage T5 Mutants Carrying Deletions in tRNA Gene Region].
- Author
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Glukhov AS, Krutilina AI, Kaliman AV, Shlyapnikov MG, and Ksenzenko VN
- Subjects
- Base Sequence, DNA, Viral genetics, Sequence Deletion, Mutation, RNA, Transfer genetics, T-Phages genetics
- Abstract
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.
- Published
- 2018
- Full Text
- View/download PDF
9. [Association of polymorphism of GSTT1 and GSTM1 genes with infertility in men].
- Author
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Kurashova NA, Belyaeva EV, Ershova OA, Dashiev BG, Bairova TA, and Kolesnikova LI
- Subjects
- Adult, Humans, Male, Russia, Base Sequence, Glutathione Transferase genetics, Infertility, Male genetics, Polymorphism, Genetic, Sequence Deletion
- Abstract
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.
- Published
- 2017
10. [Optimal Artificial Mini-Introns for Transgenic Expression in the Cells of Mice and Hamsters].
- Author
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Tikhonov MV, Maksimenko OG, Georgiev PG, and Korobko IV
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- Animals, Animals, Genetically Modified, Base Sequence, CHO Cells, Cricetulus, DNA, Complementary genetics, DNA, Complementary metabolism, Exons, Mice, Nucleotides genetics, Nucleotides metabolism, RNA, Messenger metabolism, Transcription, Genetic, Alternative Splicing, Genetic Engineering methods, Introns, RNA, Messenger genetics, Transgenes
- Abstract
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.
- Published
- 2017
- Full Text
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11. [CRISPR/CAS9, the King of Genome Editing Tools].
- Author
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Bannikov AV and Lavrov AV
- Subjects
- Animals, Bacterial Proteins metabolism, Base Pairing, Base Sequence, CRISPR-Associated Protein 9, DNA End-Joining Repair, Endonucleases metabolism, Humans, Protein Engineering, RNA, Guide, CRISPR-Cas Systems metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Recombinational DNA Repair, Bacterial Proteins genetics, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats, Endonucleases genetics, Gene Editing methods, Genome, RNA, Guide, CRISPR-Cas Systems genetics
- Abstract
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.
- Published
- 2017
- Full Text
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12. Isolating and confirming the Mudrinserted flanking sequences of maize.
- Author
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Yang WF, Tian YH, Wang TT, Wang RN, and Tao YS
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- Base Sequence, Crosses, Genetic, DNA Primers genetics, DNA Primers metabolism, Genotype, Polymerase Chain Reaction, Zea mays metabolism, DNA Transposable Elements, Gene Expression Regulation, Plant, Genes, Plant, Mutagenesis, Insertional methods, Zea mays genetics
- Abstract
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.
- Published
- 2017
13. A novel Alui-polymorphism in the fourth intron of the chicken growth hormone gene.
- Author
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Kulibaba RA, Liashenko YV, and Yurko PS
- Subjects
- Alleles, Animals, Base Sequence, Breeding, Chickens anatomy & histology, Chickens classification, Eggs, Female, Gene Expression, Gene Frequency, Genotype, Male, Meat, Chickens genetics, Deoxyribonucleases, Type II Site-Specific chemistry, Growth Hormone genetics, Introns, Polymorphism, Restriction Fragment Length, Quantitative Trait, Heritable
- Abstract
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.
- Published
- 2017
14. Establishment of transgenic lettuce plants producing potentially anti-hypertensive shRNA sequencing data.
- Author
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Gerasymenko IM, Kleschevnikov VV, Kedlian VR, Sakhno LO, Arbuzova IA, Sheludko YV, Dosenko VE, and Kuchuk NV
- Subjects
- Agrobacterium metabolism, Animals, Antihypertensive Agents chemistry, Antihypertensive Agents pharmacology, Base Pairing, Base Sequence, Binding Sites, Genetic Vectors chemistry, Genetic Vectors metabolism, Humans, Hypertension enzymology, Hypertension genetics, Hypertension pathology, Hypertension therapy, MicroRNAs genetics, MicroRNAs metabolism, Molecular Targeted Therapy, Plants, Genetically Modified, Protein Kinase C-delta antagonists & inhibitors, Protein Kinase C-delta metabolism, RNA, Small Interfering metabolism, Transformation, Genetic, Agrobacterium genetics, Antihypertensive Agents metabolism, Genetic Engineering methods, Lactuca genetics, Protein Kinase C-delta genetics, RNA, Small Interfering genetics
- Abstract
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.
- Published
- 2017
15. [Polymorphisms of KITLG, SPRY4, and BAK1 genes in patients with testicular germ cell tumors and individuals with infertility associated with AZFc deletion of the Y chromosome].
- Author
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Nemtsova MV, Ivkin EV, Simonova OA, Rudenko VV, Chernykh VB, Mikhaylenko DS, and Loran OB
- Subjects
- Adult, Humans, Male, Middle Aged, Base Sequence, Chromosomes, Human, Y genetics, Genetic Loci, Infertility, Male genetics, Intracellular Signaling Peptides and Proteins genetics, Neoplasm Proteins genetics, Neoplasms, Germ Cell and Embryonal genetics, Nerve Tissue Proteins genetics, Polymorphism, Genetic genetics, Sequence Deletion, Stem Cell Factor genetics, Testicular Neoplasms genetics, bcl-2 Homologous Antagonist-Killer Protein genetics
- Abstract
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.
- Published
- 2016
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16. Principles of creating biotransducers based on nucleic acid liquid crystals
- Author
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Iu M, Evdokimov, S G, Skuridin, V I, Salianov, V K, Rybin, and M, Palumbo
- Subjects
Cross-Linking Reagents ,Base Sequence ,DNA, Superhelical ,Nucleic Acids ,Biosensing Techniques ,Crystallization - Abstract
A brief concept of biosensors is presented. Structural peculiarities and properties of single- and double-stranded nucleic acids that are to be taken into account when creating sensing units for biosensors using different principles of molecular recognition are considered. General schemes of sensing units based on liquid-crystalline dispersions formed from low-molecular mass DNA, its complexes with "cross-linking" agents and from circular superhelical DNA molecules are described.
- Published
- 1990
17. Chromosomal DNA balance in human stem cell line 4BL.
- Author
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Akopyan HR, Kushniruk VO, Mykytenko DO, Huleuk NL, Kremenskaya Y, and Lukash LL
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- Cell Line, Chromosome Banding, Comparative Genomic Hybridization, Humans, In Situ Hybridization, Fluorescence, Karyotyping, Stem Cells pathology, Base Sequence, Chromosome Duplication, Monosomy, Ploidies, Sequence Deletion, Stem Cells metabolism
- Abstract
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.
- Published
- 2016
18. [CHARACTERISTICS OF BIOLOGICAL AND MOLECULAR-GENETIC PROPERTIES OF LACTOBACILLUS FERMENTUM 90 TC-4 PROBIOTIC STRAIN].
- Author
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Tochilina AG, Belova IV, Solovieva IV, Gorlova IS, Ivanova TP, and Zhirnov VA
- Subjects
- Base Sequence, Genome, Bacterial, High-Throughput Nucleotide Sequencing, Humans, Limosilactobacillus fermentum classification, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Limosilactobacillus fermentum genetics, Phylogeny, Probiotics, RNA, Ribosomal, 16S genetics
- Abstract
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.
- Published
- 2016
19. [Phylogenetic Analysis of Ukrainian Isolate of Beet Necrotic Yellow Vein Virus].
- Author
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Hrynchuk KV, Antipov IA, Parii MF, and Kyrychenko AM
- Subjects
- Amino Acid Sequence, Base Sequence, Ukraine, Beta vulgaris virology, Phylogeny, Plant Diseases virology, Plant Viruses classification
- Abstract
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.
- Published
- 2016
20. [Phylogenetic Analysis of Gene 16S rRNA Nucleotide Sequence of Probiotic Strain Lactobacillus sp. 55].
- Author
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Ogirchuk KS and Kovalenko NK
- Subjects
- Base Sequence, DNA, Bacterial genetics, RNA, Ribosomal, 16S genetics, Sequence Analysis, DNA, Lactobacillus genetics, Phylogeny, Probiotics
- Abstract
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.
- Published
- 2016
21. [UNIFICATION OF THE MOLECULAR EPIDEMIOLOGICAL RESEARCH OF THE TICK-BORNE ENCEPHALITIS].
- Author
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Kovalev SY and Mukhacheva TA
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Encephalitis Viruses, Tick-Borne classification, Encephalitis Viruses, Tick-Borne genetics, Encephalitis, Tick-Borne prevention & control, Encephalitis, Tick-Borne transmission, Encephalitis, Tick-Borne virology, Epidemiological Monitoring, Genetic Markers, Humans, Molecular Epidemiology, Molecular Sequence Data, Phylogeny, Russia epidemiology, Terminology as Topic, Arachnid Vectors virology, Encephalitis Viruses, Tick-Borne isolation & purification, Encephalitis, Tick-Borne epidemiology, Genes, Viral, Genome, Viral, Ticks virology
- Abstract
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.
- Published
- 2016
22. [MspI-POLYMORPHISM IN FOURTH INTRON OF THE GROWTH HORMONE GENE IN CHICKEN POPULATIONS OF DIFFERENT BREEDS. ANALYSIS OF THE CAUSES OF ADDITIONAL RESTRICTION PATTERN ORIGIN].
- Author
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Kulibaba RA, Yurko PS, and Liashenkon YV
- Subjects
- Animals, Base Sequence, Breeding, Electrophoresis, Agar Gel, Gene Frequency, Molecular Sequence Data, Nucleic Acid Heteroduplexes genetics, Polymerase Chain Reaction, Chickens genetics, Deoxyribonuclease HpaII metabolism, Growth Hormone genetics, Introns, Polymorphism, Restriction Fragment Length
- Abstract
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.
- Published
- 2015
23. [Identification of the Gene Encoding Nucleostemin in the Eye Tissues of Pleurodeles waltl].
- Author
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Markitantova YV, Avdonin PP, and Grigoryan EN
- Subjects
- Animals, Base Sequence, Molecular Sequence Data, Pleurodeles, Amphibian Proteins genetics, Amphibian Proteins metabolism, Eye Proteins genetics, Eye Proteins metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism, Retinal Pigment Epithelium metabolism
- Abstract
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.
- Published
- 2015
24. [Fish growth-hormone genes: functionality evidence of paralogous genes in Levanidov's charr].
- Author
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Kamenskaya DN, Pankova MV, Atopkin DM, and Brykov VA
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Escherichia coli genetics, Escherichia coli metabolism, Exons, Fish Proteins chemistry, Fish Proteins metabolism, Gene Expression, Growth Hormone chemistry, Growth Hormone metabolism, Introns, Molecular Sequence Data, Protein Binding, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Isoforms metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Analysis, DNA, Sequence Homology, Nucleic Acid, Transcription Factor Pit-1 genetics, Transcription Factor Pit-1 metabolism, Trout metabolism, Fish Proteins genetics, Growth Hormone genetics, Open Reading Frames, Promoter Regions, Genetic, Trout genetics
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
25. [Identification of new conserved and variable regions in the 16S rRNA gene of acetic acid bacteria and acetobacteraceae family].
- Author
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Chakravorty S, Sarkar S, and Gachhui R
- Subjects
- Acetic Acid metabolism, Acetobacteraceae classification, Acetobacteraceae metabolism, Base Sequence, Chromosome Mapping, Conserved Sequence, Genetic Variation, Molecular Sequence Data, Phylogeny, Sequence Alignment, Acetobacteraceae genetics, Genes, Bacterial, RNA, Bacterial genetics, RNA, Ribosomal, 16S genetics
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
26. [An intriguing model for 5S rDNA sequences dispersion in the genome of freshwater stingray Potamotrygon motoro (Chondrichthyes: Potamotrygonidae)].
- Author
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Cruz VP, Oliveira C, and Foresti F
- Subjects
- Animals, Base Sequence, Chromosome Mapping, Cloning, Molecular, DNA, Intergenic, Microsatellite Repeats, Molecular Sequence Data, Sequence Alignment, Sequence Analysis, DNA, Genes, rRNA, Genome, RNA, Ribosomal, 5S genetics, Skates, Fish genetics
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
27. [Identification of Hydrocarbon-Oxidizing Dietzia Bacteria from Petroleum Reservoirs Based on Phenotypic Properties and Analysis of the 16S rRNA and gyrB Genes].
- Author
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Nazina TN, Shumkova ES, Sokolova DSh, Babich TL, Zhurina MV, Xue YF, Osipov GA, Poltaraus AB, and Tourova TP
- Subjects
- Actinomycetales classification, Actinomycetales isolation & purification, Actinomycetales metabolism, Base Composition, Base Sequence, Cell Wall chemistry, DNA Gyrase genetics, Fatty Acids analysis, Molecular Sequence Data, Mycolic Acids analysis, Oxidation-Reduction, Sequence Homology, Nucleic Acid, Actinomycetales genetics, Genes, Bacterial, Hydrocarbons metabolism, Petroleum microbiology, Phylogeny, RNA, Ribosomal, 16S genetics
- Abstract
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.
- Published
- 2015
28. [The complete mitochondrial genome of peacock sole Pardachirus pavoninus (Pleuronectiformes: Soleidae) and comparative analysis of the control region among 13 soles].
- Author
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Gong L, Shi W, Si LZ, Wang ZM, and Kong XY
- Subjects
- Animals, Base Sequence, Chromosome Mapping, Conserved Sequence, Flatfishes classification, Genes, rRNA, Molecular Sequence Data, Phylogeny, Tandem Repeat Sequences, Flatfishes genetics, Genes, Mitochondrial, Genome, Mitochondrial, Locus Control Region, Mitochondria genetics
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
29. [POSSIBILITY OF USING NESTED POLYMERASE CHAIN REACTION FOR DIAGNOS- TICS OF DISEASES CAUSED BY VARICELLA ZOSTER VIRUS].
- Author
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Fam KhF, Borovikova EA, Sidorov AV, Karataeva AV, Antonova TP, and Zverev VV
- Subjects
- Base Sequence, Chickenpox genetics, Chickenpox virology, DNA, Viral isolation & purification, Herpes Zoster genetics, Herpes Zoster virology, Herpesvirus 3, Human genetics, Herpesvirus 3, Human pathogenicity, Humans, Chickenpox diagnosis, Herpes Zoster diagnosis, Herpesvirus 3, Human isolation & purification, Polymerase Chain Reaction methods
- Abstract
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.
- Published
- 2015
30. [The amplification of CYP9 genes as a preadaptation of the black garden ant Lasius niger to urban conditions].
- Author
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Konorov EA and Nikitin MA
- Subjects
- Animals, Ants classification, Ants enzymology, Base Sequence, Cities, Cytochrome P-450 Enzyme System metabolism, Fusarium chemistry, Gene Duplication, Molecular Sequence Annotation, Molecular Sequence Data, Mycotoxins metabolism, Phylogeny, Social Behavior, Xenobiotics metabolism, Adaptation, Physiological genetics, Ants genetics, Cytochrome P-450 Enzyme System genetics, Genome, Insect, Inactivation, Metabolic genetics
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
31. [Conserved motifs in the primary and secondary ITS1 structures in bryophytes].
- Author
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Milyutina IA and Ignatov MS
- Subjects
- Base Sequence, Bryophyta classification, Conserved Sequence, DNA, Ribosomal Spacer genetics, Hepatophyta classification, Molecular Sequence Data, Nucleic Acid Conformation, Phylogeny, RNA Precursors genetics, RNA, Ribosomal, 18S genetics, Saccharomyces cerevisiae genetics, Bryophyta genetics, DNA, Ribosomal Spacer chemistry, Hepatophyta genetics, RNA, Ribosomal, 18S chemistry
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
32. [Bacteriophage λ: electrostatic properties of the genome and its elements].
- Author
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Krutinina GG, Krutinin EA, Kamzolova SG, and Osypov AA
- Subjects
- Bacteriophage lambda chemistry, Base Sequence, DNA, Viral chemistry, DNA-Directed RNA Polymerases genetics, Escherichia coli genetics, Escherichia coli virology, Integrases genetics, Microbial Interactions genetics, Molecular Sequence Data, Operator Regions, Genetic, Promoter Regions, Genetic, Recombination, Genetic, Terminator Regions, Genetic, Bacteriophage lambda genetics, DNA, Viral genetics, Genome, Viral, Static Electricity
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
33. [Insertional mutation in the AZOBR_p60120 gene is accompanied by defects in the synthesis of lipopolysaccharide and calcofluor-binding polysaccharides in the bacterium Azospirillum brasilense Sp245].
- Author
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Katsy EI and Prilipov AG
- Subjects
- Base Sequence, Benzenesulfonates chemistry, Molecular Sequence Data, Azospirillum brasilense genetics, Azospirillum brasilense metabolism, Genes, Bacterial physiology, Lipopolysaccharides biosynthesis, Lipopolysaccharides genetics
- Abstract
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_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_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_p6 plasmid, encompassing the AZOBR_p60120 gene and a number of other loci, novel data on the structure of AZOBR_p6 were obtained: an approximately 5-kb gap (GenBank accession no. KM189439) was closed in the nucleotide sequence of this plasmid.
- Published
- 2015
34. [THE MICROSPORIDIUM GLUGEA GASTEROSTEI VORONIN 1974 (MICROSPORIDIA: MARINOSPORIDIA) FROM THE THREE-SPINED STICKLEBACK GASTEROSTEUS ACULEATUS (ACTINOPTERYGII: GASTEROSTEIFORMES) AS AN INDEPENDENT SPECIES].
- Author
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Tokarev YS, Voronin VN, Senderskiy IV, and Issi IV
- Subjects
- Animals, Base Sequence, Genetic Speciation, Glugea genetics, Glugea ultrastructure, Microsporidiosis microbiology, Molecular Sequence Data, RNA, Ribosomal genetics, Spores, Fungal ultrastructure, Fish Diseases microbiology, Genes, rRNA, Glugea classification, Microsporidiosis veterinary, Phylogeny, Smegmamorpha microbiology
- Abstract
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.
- Published
- 2015
35. [Opine biosynthesis and catabolism genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes].
- Author
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Vladimirov IA, Matveeva TV, and Lutova LA
- Subjects
- Agrobacterium tumefaciens metabolism, Amino Acids metabolism, Base Sequence, Ketones metabolism, Plants genetics, Polysaccharides metabolism, Agrobacterium tumefaciens genetics, Amino Acids genetics, DNA, Bacterial genetics, Plasmids genetics
- Abstract
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.
- Published
- 2015
36. [Phylogenetic analysis of Pleurotus species].
- Author
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Shnyreva AA and Shnyreva AV
- Subjects
- Base Sequence, Biological Evolution, Genetic Variation, Pleurotus genetics, Species Specificity, DNA, Ribosomal genetics, DNA, Ribosomal Spacer genetics, Phylogeny, Reproductive Isolation
- Abstract
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.
- Published
- 2015
37. [Large tandem repeats of mesocricetus a uratus in silico and in situ].
- Author
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Miheev DY, Podgornaya OI, and Ostromyshenskii DI
- Subjects
- Animals, Base Sequence, Computational Biology, Cricetinae, In Situ Hybridization, Fluorescence, Mice, Genome, Mesocricetus genetics, Tandem Repeat Sequences genetics
- Abstract
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.
- Published
- 2015
38. [Tandem repeats in rodents genome and their mapping].
- Author
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Ostromyshenskii DI, Kuznetsova LS, Komissarov AS, Kartavtseva IV, and Podgornaya L
- Subjects
- Animals, Base Sequence, Chromosome Mapping, Chromosomes, In Situ Hybridization, Fluorescence, Mice, Rodentia genetics, Species Specificity, Biological Evolution, DNA, Satellite genetics, Genome, Tandem Repeat Sequences genetics
- Abstract
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.
- Published
- 2015
39. [Methylation status of line-1 retrotransposon in chromosomal mosaicism during the early stages of human embryonic development].
- Author
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Vasil'ev SA, Tolmacheva EN, Kashevarova AA, Sazhenova EA, and Lebedev IN
- Subjects
- Abortion, Spontaneous genetics, Abortion, Spontaneous pathology, Aneuploidy, Base Sequence, Chromosome Aberrations, Female, Humans, Pregnancy, DNA Methylation genetics, Embryonic Development genetics, Long Interspersed Nucleotide Elements genetics, Mosaicism
- Abstract
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.
- Published
- 2015
40. [CITRULLINUREIDASE GENE DIVERSITY IN THE GENUS FRANCISELLA].
- Author
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Timofeev VS, Bakhteeva IV, Pavlov VM, and Mokrievich AN
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Base Sequence, Francisella enzymology, Genetic Variation, Molecular Sequence Data, Urease chemistry, Urease metabolism, Bacterial Proteins genetics, Citrulline metabolism, Francisella genetics, Phylogeny, Urease genetics
- Abstract
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_0827 encoding the C-N hydrolase, which probably provides the citrullinureidase activity.
- Published
- 2015
41. [The molecular genetic characteristic of RNA of human enterovirus detected in bio-test from child with serous meningitis].
- Author
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Onishchenko GG, Petrov AA, Kazantsev AV, Suroviatkin AV, Kokonova MS, Lebedev VN, Alekseev IaI, Varlamov DA, Kutaev DA, Vakhnov EIu, and Borisevich SV
- Subjects
- Base Sequence, China, Diagnosis, Enterovirus A, Human genetics, Enterovirus Infections diagnosis, Enterovirus Infections genetics, Enterovirus Infections mortality, Humans, Meningitis mortality, Meningitis virology, Phylogeny, RNA, Viral genetics, Russia, Enterovirus A, Human isolation & purification, Enterovirus Infections virology, RNA, Viral isolation & purification
- Abstract
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.
- Published
- 2014
42. [Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus].
- Author
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Slugina MA, Torres Minho K, and Filiushin MA
- Subjects
- Base Sequence, Evolution, Molecular, Genetic Markers, Genetics, Population, Molecular Sequence Data, Mutagenesis, Insertional, Phylogeny, Polymorphism, Single Nucleotide, Sequence Analysis, DNA, Sequence Deletion, Species Specificity, Amaranthus classification, Amaranthus genetics, DNA, Ribosomal Spacer genetics, Genes, rRNA, Genetic Speciation, RNA, Ribosomal, 5.8S genetics
- Abstract
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.
- Published
- 2014
43. [Analysis of nucleotide sequences of cytochrome C oxidase I (CO1) gene of mtDNA of Arctic Liopsetta glacialis and striped Liopsetta pinnifasciata flounder (Pleuronectidae) of the Sea of Okhotsk].
- Author
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Potapova NA, Pustovoĭt SP, and Iusupov RR
- Subjects
- Animals, Base Sequence, Cold Climate, Data Interpretation, Statistical, Flatfishes growth & development, Haplotypes, Molecular Sequence Data, Muscle, Skeletal enzymology, Oceans and Seas, Seasons, Sequence Homology, Nucleic Acid, Species Specificity, DNA, Mitochondrial genetics, Electron Transport Complex IV genetics, Flatfishes genetics
- Abstract
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.
- Published
- 2014
44. [Cloning and analysis of a new aliphatic amidase gene from Rhodococcus erythropolis TA37].
- Author
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Lavrov KV, Karpova IY, Epremyan AS, and Yanenko AS
- Subjects
- ATP-Binding Cassette Transporters genetics, Acetamides metabolism, Acetanilides metabolism, Amidohydrolases metabolism, Amino Acid Sequence, Bacterial Proteins metabolism, Base Sequence, Cloning, Molecular, Coenzyme A Ligases genetics, Genes, Bacterial, Molecular Sequence Data, Rhodococcus enzymology, Substrate Specificity, Amidohydrolases genetics, Bacterial Proteins genetics, Rhodococcus genetics
- Abstract
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.
- Published
- 2014
45. [cDNA library construction from panicle meristem of finger millet].
- Author
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Radchuk V, Pirko IaV, Isaenkov SV, Emets AI, and Blium IaB
- Subjects
- Base Sequence, Cloning, Molecular, DNA Primers genetics, DNA, Plant isolation & purification, Escherichia coli genetics, Molecular Sequence Data, RNA, Plant genetics, RNA, Plant isolation & purification, DNA, Complementary genetics, DNA, Plant genetics, Eleusine genetics, Gene Library, Meristem genetics
- Abstract
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.
- Published
- 2014
46. [Molecular cloning, structural analysis, and expression of zona pellucida glycoprotein ZP3 gene from Chinese zokor, Myospalax fontanierii].
- Author
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Sui DD, Wu JL, Zhang H, Li H, Zhou ZM, Zhang DH, and Han CX
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, Blotting, Western, China, Cloning, Molecular, Egg Proteins chemistry, Escherichia coli genetics, Female, Gene Expression Regulation, Histidine genetics, Membrane Glycoproteins chemistry, Mice, Molecular Sequence Data, Open Reading Frames, Phylogeny, Rats, Receptors, Cell Surface chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins isolation & purification, Rodentia, Sequence Homology, Amino Acid, Zona Pellucida Glycoproteins, Egg Proteins genetics, Egg Proteins metabolism, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism, Receptors, Cell Surface genetics, Receptors, Cell Surface metabolism
- Abstract
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.
- Published
- 2014
- Full Text
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47. [Identification of hypervariable regions within the 16S-23S rRNA intergenic spacer region of Flavobacterium columnare and its application in assigning genomovar group to an individual strain].
- Author
-
Rathore G and Verma DK
- Subjects
- Base Sequence, DNA Barcoding, Taxonomic, Flavobacterium classification, Molecular Sequence Data, Phylogeny, RNA, Ribosomal, 16S, RNA, Ribosomal, 23S, Sequence Homology, Nucleic Acid, DNA, Intergenic, DNA, Ribosomal, Flavobacterium genetics, Genetic Variation
- Abstract
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.
- Published
- 2014
48. [The effect of topology of quorum sensing-related genes in Pectobacterium atrosepticumon their expression].
- Author
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Gogoleva NE, Shlykova LV, Gorshkov VIu, Daminova AG, and Gogolev IuV
- Subjects
- Acyl-Butyrolactones metabolism, Base Sequence, Molecular Sequence Data, Pectobacterium genetics, Gene Expression Regulation, Bacterial, Pectobacterium physiology, Quorum Sensing genetics
- Abstract
In prokaryotic genomes, the neighboring genes are often located on the complementary DNA strands and adjoin each other by their 5'- or 3'-ends or even overlap by their open reading frames. It was suggested that such gene topology hasfunctional purpose providing the regulation of their expression. For those genes that overlap by their coding 3'-termini this assumption has not been confirmed experimentally. In a broad group of bacteria that belong to proteobacteria such a convergent gene arrangement is typical for functionally connected quorum sensing-related genes "P" and "R" that encode synthases of N-acyl homoserine lactones and their sensors, respectively. In the present study on the example of overlapping quorum sensing-related genes of plant pathogenic bacterium Pectobacterium atrosepticum SCRI1043--expI and expR it was shown that the topology of these genes determines the regula- tion of their expression.
- Published
- 2014
49. [Polymorphism among RFL-PPR homologs in sunflower (Helianthus annuus L.) lines with varying ability for the suppression of the cytoplasmic male sterility phenotype].
- Author
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Anisimova IN, Alpatieva NV, Rozhkova VT, Kuznetsova EB, Pinaev AG, and Gavrilova VA
- Subjects
- Base Sequence, Expressed Sequence Tags, Genes, Plant, Helianthus physiology, Introns, Molecular Sequence Data, Plant Proteins genetics, Transcription Factors genetics, Helianthus genetics, Plant Infertility genetics, Pollen genetics, Polymorphism, Genetic
- Abstract
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.
- Published
- 2014
50. [An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics].
- Author
-
Dmitrochenko AE, Turiianskaia OM, Gilep AA, Usanov SA, and Iantsevich AV
- Subjects
- Amino Acid Sequence, Base Sequence, Escherichia coli K12 enzymology, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Gene Expression, Hot Temperature, Hydrogen-Ion Concentration, Indicators and Reagents, Kinetics, Mass Spectrometry, Molecular Sequence Data, Plasmids metabolism, Protein Stability, Protein Unfolding, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Thermodynamics, Uracil-DNA Glycosidase chemistry, Uracil-DNA Glycosidase metabolism, Escherichia coli K12 genetics, Escherichia coli Proteins genetics, Plasmids chemistry, Polymerase Chain Reaction standards, Recombinant Fusion Proteins genetics, Uracil-DNA Glycosidase genetics
- Abstract
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.
- Published
- 2014
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