Tatyana L. Azhikina


PeriodCountry, cityEducation institutionAdditional info
1984 Russia, Moscow M.V. Lomonosov Moscow State University (Chemical faculty) Ms in organic chemistry
1991 Russia, Moscow Shemyakin Institute of Bioorganic Chemisrty PhD in chemistry, specialization: bioorganic chemistry (thesis: Synthesis, cloning and expression of human inteleukin-3 gene)
2009 Russia, Moscow Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences DSc in biology, specialization: molecular biology (thesis: Hybridization approaches in large-scale studies of genomes)

Selected publications

  1. Gainetdinov I., Skvortsova Y., Kondratieva S., Funikov S., Azhikina T. (2017). Two modes of targeting transposable elements by piRNA pathway in human testis. RNA 23 (11), 1614–1625 [+]

    PIWI proteins and their partner small RNAs, termed piRNAs, are known to control transposable elements (TEs) in the germline. Here, we provide evidence that in humans this control is exerted in two different modes. On the one hand, production of piRNAs specifically targeting evolutionarily youngest TEs (L1HS, L1PA2-L1PA6, LTR12C, SVA) is present both at prenatal and postnatal stages of spermatogenesis and is performed without involvement of piRNA clusters. On the other hand, at postnatal stages, piRNAs deriving from pachytene clusters target "older" TEs and thus complement cluster-independent piRNA production to achieve relevant targeting of virtually all TEs expressed in postnatal testis. We also find that converging transcription of antisense-oriented genes contributes to the origin of genic postnatal prepachytene clusters. Finally, while a fraction of pachytene piRNAs was previously shown to arise from long intergenic noncoding RNAs (lincRNAs, i.e., pachytene piRNA cluster primary transcripts), we ascertain that these are a specific set of lincRNAs that both possess distinguishing epigenetic features and are expressed exclusively in testis.

  2. Boyko A.A., Azhikina T.L., Streltsova M.A., Sapozhnikov A.M., Kovalenko E.I. (2016). HSP70 in human polymorphonuclear and mononuclear leukocytes: comparison of the protein content and transcriptional activity of HSPA genes. Cell Stress Chaperones , [+]

    Cell-type specific variations are typical for the expression of different members of the HSP70 family. In circulating immune cells, HSP70 proteins interact with units of signaling pathways involved in the immune responses and may promote cell survival in sites of inflammation. In this work, we compared basal HSP70 expression and stress-induced HSP70 response in polymorphonuclear and mononuclear human leukocytes. The intracellular content of inducible and constitutive forms of HSP70 was analyzed in relation to the transcriptional activity of HSPA genes. Hyperthermia was used as the stress model for induction of HSP70 synthesis in the cells. Our results demonstrated that granulocytes (mainly neutrophils) and mononuclear cells differ significantly by both basal HSP70 expression and levels of HSP70 induction under hyperthermia. The differences were observed at the levels of HSPA gene transcription and intracellular HSP70 content. The expression of constitutive Hsс70 protein was much higher in mononuclear cells consisting of monocytes and lymphocytes than in granulocytes. At the same time, intact neutrophils showed increased expression of inducible Hsp70 protein compared to mononuclear cells. Heat treatment induced additional expression of HSPA genes in leukocytes. The most pronounced increase in the expression was observed in polymorphonuclear and mononuclear leukocytes for HSPA1A/B. However, in granulocytes, the induction of the transcription of the HSPA8 gene encoding the Hsc70 protein was significantly higher than in mononuclear cells. These variations in transcriptional activity of HSPA genes and intracellular HSP70 content in different populations of leukocytes may reflect specified requirements for the chaperone activity in the cells with a distinct functional role in the immune system.

  3. Gainetdinov I.V., Kapitskaya K.Y., Rykova E.Y., Ponomaryova A.A., Cherdyntseva N.V., Vlassov V.V., Laktionov P.P., Azhikina T.L. (2016). Hypomethylation of human-specific family of LINE-1 retrotransposons in circulating DNA of lung cancer patients. Lung Cancer 99, 127–30 [+]

    Circulating DNA has recently gained attention as a fast and non-invasive way to assess tumor biomarkers. Since hypomethylation of LINE-1 repetitive elements was described as one of the key hallmarks of tumorigenesis, we aimed to establish whether the methylation level of LINE-1 retrotransposons changes in cell-surface-bound fraction of circulating DNA (csbDNA) of lung cancer patients. Methylated CpG Island Recovery Assay (MIRA) coupled to qPCR-based quantitation was performed to assess integral methylation level of LINE-1 promoters in csbDNA of non-small cell lung cancer patients (n=56) and healthy controls (n=44). Deep sequencing of amplicons revealed that hypomethylation of LINE-1 promoters in csbDNA of lung cancer patients is more pronounced for the human-specific L1Hs family. Statistical analysis demonstrates significant difference in LINE-1 promoter methylation index between cancer patients and healthy individuals (ROC-curve analysis: n=100, AUC=0.69, p=0.0012) and supports the feasibility of MIRA as a promising non-invasive approach.

  4. Gainetdinov I.V., Kondratieva S.A., Skvortsova Y.V., Zinovyeva M.V., Stukacheva E.A., Klimov A., Tryakin A.A., Azhikina T.L. (2016). Distinguishing epigenetic features of preneoplastic testis tissues adjacent to seminomas and nonseminomas. Oncotarget , [+]

    PIWI pathway proteins are expressed during spermatogenesis where they play a key role in germ cell development. Epigenetic loss of PIWI proteins expression was previously demonstrated in testicular germ cell tumors (TGCTs), implying their involvement in TGCT development. In this work, apart from studying only normal testis and TGCT samples, we also analyzed an intermediate stage, i.e. preneoplastic testis tissues adjacent to TGCTs. Importantly, in this study, we minimized the contribution of patient-to-patient heterogeneity by using matched preneoplastic/TGCT samples. Surprisingly, expression of germ cell marker DDX4 suggests that spermatogenesis is retained in premalignant testis tissues adjacent to nonseminoma, but not those adjacent to seminoma. Moreover, this pattern is followed by expression of PIWI pathway genes, which impacts one of their functions: DNA methylation level over LINE-1 promoters is higher in preneoplastic testis tissues adjacent to nonseminomas than those adjacent to seminomas. This finding might imply distinct routes for development of the two types of TGCTs and could be used as a novel diagnostic marker, possibly, noninvasively. Finally, we studied the role of CpG island methylation in expression of PIWI genes in patient samples and using in vitro experiments in cell line models: a more complex interrelation between DNA methylation and expression of the corresponding genes was revealed.

  5. Skvortsova Y.V., Kondratieva S.A., Zinovyeva M.V., Nikolaev L.G., Azhikina T.L., Gainetdinov I.V. (2016). Intragenic Locus in Human PIWIL2 Gene Shares Promoter and Enhancer Functions. PLoS ONE 11 (6), e0156454 [+]

    Recently, more evidence supporting common nature of promoters and enhancers has been accumulated. In this work, we present data on chromatin modifications and non-polyadenylated transcription characteristic for enhancers as well as results of in vitro luciferase reporter assays suggesting that PIWIL2 alternative promoter in exon 7 also functions as an enhancer for gene PHYHIP located 60Kb upstream. This finding of an intragenic enhancer serving as a promoter for a shorter protein isoform implies broader impact on understanding enhancer-promoter networks in regulation of gene expression.

  6. Azhikina T.L., Ignatov D.V., Salina E.G., Fursov M.V., Kaprelyants A.S. (2015). Role of Small Noncoding RNAs in Bacterial Metabolism. Biochemistry Mosc. 80 (13), 1633–46 [+]

    The study of prokaryotic small RNAs is one of the most important directions in modern molecular biology. In the last decade, multiple short regulatory transcripts have been found in prokaryotes, and for some of them functional roles have been elucidated. Bacterial small RNAs are implicated in the regulation of transcription and translation, and they affect mRNA stability and gene expression via different mechanisms, including changes in mRNA conformation and interaction with proteins. Most small RNAs are expressed in response to external factors, and they help bacteria to adapt to changing environmental conditions. Bacterial infections of various origins remain a serious medical problem, despite significant progress in fighting them. Discovery of mechanisms that bacteria employ to survive in infected organisms and ways to block these mechanisms is promising for finding new treatments for bacterial infections. Regulation of pathogenesis with small RNAs is an attractive example of such mechanisms. This review considers the role of bacterial small RNAs in adaptation to stress conditions. We pay special attention to the role of small RNAs in Mycobacterium tuberculosis infection, in particular during establishment and maintenance of latent infection.

  7. Ignatov D.V., Salina E.G., Fursov M.V., Skvortsov T.A., Azhikina T.L., Kaprelyants A.S. (2015). Dormant non-culturable Mycobacterium tuberculosis retains stable low-abundant mRNA. BMC Genomics 16 (1), 954 [+]

    Dormant Mycobacterium tuberculosis bacilli are believed to play an important role in latent tuberculosis infection. Previously, we have demonstrated that cultivation of M. tuberculosis in K(+)-deficient medium resulted in generation of dormant cells. These bacilli were non-culturable on solid media (a key feature of dormant M. tuberculosis in vivo) and characterized by low metabolism and tolerance to anti-tuberculosis drugs. The dormant bacteria demonstrated a high potential to reactivation after K(+) reintroduction even after prolonged persistence under rifampicin. In this work, we studied the transcriptome and stability of transcripts in persisting dormant bacilli under arrest of mRNA de novo synthesis.

  8. Kovalenko E.I., Boyko A.A., Semenkov V.F., Lutsenko G.V., Grechikhina M.V., Kanevskiy L.M., Azhikina T.L., Telford W.G., Sapozhnikov A.M. (2014). ROS production, intracellular HSP70 levels and their relationship in human neutrophils: effects of age. Oncotarget 5 (23), 11800–12 [+]

    ROS production and intracellular HSP70 levels were measured in human neutrophils for three age groups: young (20-59 years), elders (60-89 years) and nonagenarians (90 years and older). Elders showed higher levels of spontaneous intracellular ROS content compared with young and nonagenarian groups, which had similar intracellular ROS levels. Zymosan-induced (non-spontaneous) extracellular ROS levels were also similar for young and nonagenarians but were lower in elders. However, spontaneous extracellular ROS production increased continuously with age. Correlation analysis revealed positive relationships between HSP70 levels and zymosan-stimulated ROS production in the elder group. This was consistent with a promoting role for HSP70 in ROS-associated neutrophils response to pathogens. No positive correlation between ROS production and intracellular HSP70 levels was found for groups of young people and nonagenarians. In contrast, significant negative correlations of some ROS and HSP70 characteriscics were found for neutrophils from young people and nonagenarians. The observed difference in ROS and HSP70 correlations in elders and nonagenarians might be associated with an increased risk of mortality in older individuals less than 90 years old.

  9. Kondratieva T., Azhikina T., Nikonenko B., Kaprelyants A., Apt A. (2014). Latent tuberculosis infection: what we know about its genetic control? Tuberculosis (Edinb) 94 (5), 462–8 [+]

    About 90% of all cases of tuberculosis (TB) infection are comprised of latent mycobacterial persistence in the absence of clinical manifestations. In a proportion of latently infected individuals infection eventually reactivates and becomes contagious, seriously influencing epidemiological situation. Mechanisms of Mycobacterium tuberculosis transition to dormancy and TB reactivation are poorly understood, and biological markers of latency remain largely unknown. Data are accumulating that the dynamical equilibrium between the parasite and the host (expressed as a long term asymptomatic infection) and its abrogation (expressed as a reactivation disease) are genetically controlled by both parties. In this short review, the authors summarize the results of experimental studies on genetic regulation of the latent TB infection.

  10. Kapitskaya K.Y., Azhikina T.L., Ponomaryova A.A., Cherdyntseva N.V., Vlasov V.V., Laktionov P.P., Rykova E.Y. (2014). MIRA analysis of RARβ2 gene methylation in DNA circulating in the blood in lung cancer. Bull. Exp. Biol. Med. 157 (4), 516–9 [+]

    Analysis of DNA epigenetic mutations in the blood circulating DNA is a prospective trend for creation of noninvasive methods for the diagnosis and treatment efficiency monitoring in cancer. The methylation status of target genes in circulating DNA was evaluated by methods based on preliminary bisulfite conversion of DNA. We used a different approach based on selection of hypermethylated sequences of circulating DNA by means of DNA-methyl-binding protein (methylated CpG island recovery assay, MIRA). Methylation was evaluated for RARβ2 tumor suppression gene in circulating DNA in lung cancer and a trend was detected to higher methylation of this gene in the patients in comparison with healthy donors.

  11. Bychenko O.S., Sukhanova L.V., Azhikina T.L., Skvortsov T.A., Belomestnykh T.V., Sverdlov E.D. (2014). Differences in brain transcriptomes of closely related baikal coregonid species. Biomed Res Int 2014, 857329 [+]

    The aim of this work was to get deeper insight into genetic factors involved in the adaptive divergence of closely related species, specifically two representatives of Baikal coregonids-Baikal whitefish (Coregonus baicalensis Dybowski) and Baikal omul (Coregonus migratorius Georgi)-that diverged from a common ancestor as recently as 10-20 thousand years ago. Using the Serial Analysis of Gene Expression method, we obtained libraries of short representative cDNA sequences (tags) from the brains of Baikal whitefish and omul. A comparative analysis of the libraries revealed quantitative differences among ~4% tags of the fishes under study. Based on the similarity of these tags with cDNA of known organisms, we identified candidate genes taking part in adaptive divergence. The most important candidate genes related to the adaptation of Baikal whitefish and Baikal omul, identified in this work, belong to the genes of cell metabolism, nervous and immune systems, protein synthesis, and regulatory genes as well as to DTSsa4 Tc1-like transposons which are widespread among fishes.

  12. Gainetdinov I.V., Skvortsova Y.V., Stukacheva E.A., Bychenko O.S., Kondratieva S.A., Zinovieva M.V., Azhikina T.L. (2014). Expression Profiles of PIWIL2 Short Isoforms Differ in Testicular Germ Cell Tumors of Various Differentiation Subtypes. PLoS ONE 9 (11), e112528 [+]

    PIWI family proteins have recently emerged as essential contributors in numerous biological processes including germ cell development, stem cell maintenance and epigenetic reprogramming. Expression of some of the family members has been shown to be elevated in tumors. In particular, PIWIL2 has been probed as a potential neoplasia biomarker in many cancers in humans. Previously, PIWIL2 was shown to be expressed in most tumours as a set of its shorter isoforms. In this work, we demonstrated the presence of its 60 kDa (PL2L60A) and 80 kDa (PL2L80A) isoforms in testicular cancer cell lines. We also ascertained the transcriptional boundaries of mRNAs and alternative promoter regions for these PIWIL2 isoforms. Further, we probed a range of testicular germ cell tumor (TGCT) samples and found PIWIL2 to be predominantly expressed as PL2L60A in most of them. Importantly, the levels of both PL2L60A mRNA and protein products were found to vary depending on the differentiation subtype of TGCTs, i.e., PL2L60A expression is significantly higher in undifferentiated seminomas and appears to be substantially decreased in mixed and nonseminomatous TGCTs. The higher level of PL2L60A expression in undifferentiated TGCTs was further validated in the model system of retinoic acid induced differentiation in NT2/D1 cell line. Therefore, both PL2L60A mRNA and protein abundance could serve as an additional marker distinguishing between seminomas and nonseminomatous tumors with different prognosis and therapy approaches.

  13. Skvortsov T.A., Ignatov D.V., Majorov K.B., Apt A.S., Azhikina T.L. (2013). Mycobacterium tuberculosis Transcriptome Profiling in Mice with Genetically Different Susceptibility to Tuberculosis. Acta Naturae 5 (2), 62–9 [+]

    Whole transcriptome profiling is now almost routinely used in various fields of biology, including microbiology. In vivo transcriptome studies usually provide relevant information about the biological processes in the organism and thus are indispensable for the formulation of hypotheses, testing, and correcting. In this study, we describe the results of genome-wide transcriptional profiling of the major human bacterial pathogen M. tuberculosis during its persistence in lungs. Two mouse strains differing in their susceptibility to tuberculosis were used for experimental infection with M. tuberculosis. Mycobacterial transcriptomes obtained from the infected tissues of the mice at two different time points were analyzed by deep sequencing and compared. It was hypothesized that the changes in the M. tuberculosis transcriptome may attest to the activation of the metabolism of lipids and amino acids, transition to anaerobic respiration, and increased expression of the factors modulating the immune response. A total of 209 genes were determined whose expression increased with disease progression in both host strains (commonly upregulated genes, CUG). Among them, the genes related to the functional categories of lipid metabolism, cell wall, and cell processes are of great interest. It was assumed that the products of these genes are involved in M. tuberculosis adaptation to the host immune system defense, thus being potential targets for drug development.

  14. Ignatov D., Malakho S., Majorov K., Skvortsov T., Apt A., Azhikina T. (2013). RNA-Seq Analysis of Mycobacterium avium Non-Coding Transcriptome. PLoS ONE 8 (9), e74209 [+]

    Deep sequencing was implemented to study the transcriptional landscape of Mycobacterium avium. High-resolution transcriptome analysis identified the transcription start points for 652 genes. One third of these genes represented leaderless transcripts, whereas the rest of the transcripts had 5' UTRs with the mean length of 83 nt. In addition, the 5' UTRs of 6 genes contained SAM-IV and Ykok types of riboswitches. 87 antisense RNAs and 10 intergenic small RNAs were mapped. 6 intergenic small RNAs, including 4.5S RNA and rnpB, were transcribed at extremely high levels. Although several intergenic sRNAs are conserved in M. avium and M. tuberculosis, both of these species have unique intergenic sRNAs. Moreover, we demonstrated that even conserved small RNAs are regulated differently in these species. Different sets of intergenic sRNAs may underlie differences in physiology between conditionally pathogenic M. avium and highly specialized pathogen M. tuberculosis.

  15. Ignatov D., Kondratieva E., Azhikina T., Apt A. (2012). Mycobacterium avium-triggered diseases: pathogenomics. Cell. Microbiol. 14 (6), 808–18 [+]

    The species Mycobacterium avium includes several subspecies representing highly specialized avian and mammalian pathogens, non-obligatory pathogens of immune compromised humans and saprophitic organisms. Recently obtained information concerning the diversity of M. avium genomic structures not only clarified phylogenic relationships within this species, but began to shed light on the question of how such closely related microorganisms adapt to the occupation of distinct ecological niches. In this review we discuss specific features of M. avium genetic composition, as well as genetic and molecular aspects of M. avium hominissuis (MAH)-triggered disease pathogenesis, including virulence, penetration, immune response manipulation and host genetic control.

  16. Bulanenkova S.S., Kozlova A.A., Kotova E.S., Snezhkov E.V., Azhikina T.L., Akopov S.B., Nikolaev L.G., Sverdlov E.D. (2011). Dam methylase accessibility as an instrument for analysis of mammalian chromatin structure. Epigenetics 6 (9), 1078–1084 [+]

    For a 140 kb human genome locus, an analysis of the distribution of Dam methylase accessible sites, DNase I sensitive and resistant regions, unmethylated CpG sites and acetylated histone H3 molecules was performed and compared with transcriptional activity of the genes within the locus. A direct correlation was found between the extent of Dam methylation and C5 cytosine (CpG) methylation. It was also demonstrated that promoter regions of all highly and moderately transcribed genes are highly accessible to methylation by Dam methylase. In contrast, promoters of non transcribed genes showed a very low extent of Dam methylation. Promoter regions of non transcribed genes were also highly CpG methylated, and the promoter and more distant 5'-regions of the housekeeping gene COX6B1 were substantially CpG demethylated. Some highly Dam methylase accessible regions are present in the intergenic regions of the locus suggesting that the latter contain either unidentified non-coding transcripts or extended regulatory elements like locus control regions.

  17. Azhikina T., Kozlova A., Skvortsov T., Sverdlov E. (2011). Heterogeneity and degree of TIMP4, GATA4, SOX18, and EGFL7 gene promoter methylation in non-small cell lung cancer and surrounding tissues. Cancer Genet 204 (9), 492–500 [+]

    We used methylation-sensitive high resolution melting analysis to assess methylation of CpG islands within the promoters of the TIMP4, GATA4, SOX18, and EGFL7 genes in samples of non-small cell lung cancer and surrounding apparently normal tissue and noncancerous lung tissues. We found that the promoter methylation was heterogeneous in both tumor and surrounding normal tissue. This is in contrast to healthy lung tissue, where the promoters were normally either non- or hypomethylated, and the heterogeneity of methylation was low. An increased heterogeneity of methylation in the normal tissues surrounding the tumor may suggest an early start of epigenetic processes preceding genetic and morphologic changes and can be used as a biomarker of early cancerization events. This analysis is an easy and sensitive tool for studying epigenetic heterogeneity and could be used in clinical practice.

  18. Ignatov D.V., Skvortsov T.A., Majorov K.B., Apt A.S., Azhikina T.L. (2010). Adaptive Changes in Mycobacterium avium Gene Expression Profile Following Infection of Genetically Susceptible and Resistant Mice. Acta Naturae 2 (3), 78–83 [+]

    We performed a comparative analysis ofMycobacterium aviumtranscriptomes (strain 724R) in infected mice of two different strains- resistant and susceptible to infection. Sets of mycobacterial genes transcribed in lung tissue were defined, and differentially transcribed genes were revealed. Our results indicate thatM. aviumgenes coding for enzymes of the Krebs cycle, oxidative phosphorylation, NO reduction, fatty acid biosynthesis, replication, translation, and genome modification are expressed at high levels in the lungs of genetically susceptible mice. The expression of genes responsible for cell wall properties, anaerobic nitrate respiration, fatty acid degradation, synthesis of polycyclic fatty acid derivatives, and biosynthesis of mycobactin and other polyketides is increased in the resistant mice. In the resistant host environment,Mycobacterium aviumapparently transitions to a latent state caused by the deficiency in divalent cations and characterised by anaerobic respiration, degradation of fatty acids, and modification of cell wall properties.

  19. Azhikina T., Skvortsov T., Radaeva T., Mardanov A., Ravin N., Apt A., Sverdlov E. (2010). A new technique for obtaining whole pathogen transcriptomes from infected host tissues. BioTechniques 48 (2), 139–44 [+]

    We propose a novel experimental approach based on coincidence cloning for analyzing sequences of bacterial intracellular pathogens specifically transcribed in affected tissues. Co-denaturation and co-renaturation of excess bacterial genomic DNA with the cDNA prepared on total RNA of the infected tissue allows one to select the bacterial fraction of the cDNA sample. We used this technique for preparing and characterizing the Mycobacterium tuberculosis cDNA pool, representing the transcriptome of infected mouse lungs in the chronic phase of infection. A cDNA pool enriched in fragments of mycobacterial cDNA was analyzed by the high-throughput 454 sequencing procedure. We demonstrated that its composition corresponded to what can be expected in the chronic phase of infection and, after the adaptation of M. tuberculosis to the host immune system, was characterized by an active lipid metabolism and switched from aerobic to anaerobic respiration. The technique is universal and requires no prior knowledge of the pathogen genome sequence. Pools of transcribed sequences obtained by this technique retain the main characteristics of the genome-wide gene transcription pattern within infected tissue, and can be used for in vivo analysis of gene expression of a wide spectrum of infection agents, such as viruses, bacteria, and protista.

  20. Skvortsova Y.V., Azhikina T.L., Stukacheva E.A., Sverdlov E.D. (2009). Studies on functional role of DNA methylation within the FXYD5-COX7A1 region of human chromosome 19. Biochemistry Mosc. 74 (8), 874–81 [+]

    We used the Rapid Identification of Genomic Splits technique to get a detailed methylation landscape of a 1-megabase-long human genome region (FXYD5-COX7A1, chromosome 19) in normal and tumor lung tissues and in the A549 lung cancer cell line. All three samples were characterized by an essentially uneven density of unmethylated sites along the fragment. Strikingly enough, the distribution of hypomethylated regions did not correlate with gene locations within the fragment. We also demonstrated that the methylation pattern of this long genomic DNA fragment was rather stable and practically unchanged in human lung cancer tissue as compared with its normal counterpart. On the other hand, the methylation landscape obtained for the A549 cell line (human lung carcinoma) in the USF2-MAG locus showed clear differences from that of the tissues mentioned above. A comparative analysis of transcriptional activity of the genes in this region demonstrated the general absence of direct correlation between methylation and expression, although some data suggest a possible role of methylation in the regulation of MAG expression through cis-regulatory elements. In total, our data provide new evidence for the necessity of revising currently prevailing views on the functional significance of methyl groups in genomic DNA.

  21. Skvortsov T.A., Azhikina T.L. (2009). [Transcriptome analysis of bacterial pathogens in vivo: problems and solutions]. Bioorg. Khim. 36 (5), 596–606 [+]

    This review considers modern strategy of whole-transcriptome investigation of intracellular pathogens in vivo. The methods of preliminary enrichment for bacterial RNA are discussed in details, including hybridization-based approaches and the peculiarities of cDNA synthesis in bacteria; methods of synthesizing cDNA from the view of features of prokaryotic RNAs and methods of bacterial cDNA analysis are also described, including high-throughput RNA-seq. The discussed methods are exemplified by analysis of Mycobacterium tuberculosis in different infection models: in cell lines, infected animal tissues and organs, and human surgical samples of lung. The advantages and limitations of different methodological approaches are discussed.

  22. Bychenko O.S., Sukhanova L.V., Ukolova S.S., Skvortsov T.A., Potapov V.K., Azhikina T.L., Sverdlov E.D. (2009). [Genome similarity of Baikal omul and sig]. Bioorg. Khim. 35 (1), 95–102 [+]

    Two members of the Baikal sig family, a lake sig (Coregonus lavaretus baicalensis Dybovsky) and omul (C. autumnalis migratorius Georgi), are close relatives that diverged from the same ancestor 10-20 thousand years ago. In this work, we studied genomic polymorphism of these two fish species. The method of subtraction hybridization (SH) did not reveal the presence of extended sequences in the sig genome and their absence in the omul genome. All the fragments found by SH corresponded to polymorphous noncoding genome regions varying in mononucleotide substitutions and short deletions. Many of them are mapped close to genes of the immune system and have regions identical to the Tc-1-like transposons abundant among fish, whose transcription activity may affect the expression of adjacent genes. Thus, we showed for the first time that genetic differences between Baikal sig family members are extremely small and cannot be revealed by the SH method. This is another endorsement of the hypothesis on the close relationship between Baikal sig and omul and their evolutionarily recent divergence from a common ancestor.

  23. Ignatov D.V., Mefodeva L.G., Maĭorov K.B., Skvortsov T.A., Azhikina T.L. (2009). [Identified small RNAs of Mycobacterium avium]. Bioorg. Khim. 38 (4), 509–12 [+]

    Posttranscriptional regulation of gene expression by small RNAs was shown for multiple pathogenic microorganisms and plays an important role in virulence. 4 putative sRNA genes located in intergenic loci were identified: MAV_0380-0381 (4.5S RNA), MAV_1034-1035 (trans-encoded sRNA), MAV_1415-1416 (antisense or trans-encoded sRNA) and MAV_1531-1532 (processed 5' UTR of 16S rRNA gene). The revealed sRNAs represent the first small noncoding RNAs identified in M. avium.

  24. Skvortsov T.A., Azhikina T.L. (2009). [Adaptive changes of Mycobacterium tuberculosis gene expression during the infectious process]. Bioorg. Khim. 38 (4), 391–405 [+]

    Mycobacterium tuberculosis causes an infection in humans with clinical manifestations varying from asymptomatic carriage of bacteria to rapidly progressing tuberculosis. Infection outcomes depend on complex and still not fully understood interactions between the pathogenic bacteria and their host organism. Gene expression changes in response to host defense mechanisms are needed for M. tuberculosis survival and functioning. This review focuses on the analysis of dynamic changes in the M. tuberculosis transcriptome taking place during infection processes in host tissues. Presently available data on mycobacterial transcriptome changes obtained from different infection models are discussed. A major part of this review is devoted to the description of biochemical changes occurring in M. tuberculosis infection process, from the primary through latent infection to pathogen reactivation. At each stage of the infection, gene expression changes and induced bacterial metabolic variations are discussed.

  25. Gainetdinov I.V., Azhikina T.L., Sverdlov E.D. (2007). Use of short representative sequences for structural and functional genomic studies. Biochemistry Mosc. 72 (11), 1179–86 [+]

    Existing approaches to direct genomic studies are costly and time-consuming. To overcome these problems, a series of tag-based methods utilizing short fragments uniquely representing full-length transcripts/genes from which they originate has been developed. This review summarizes basic principles underlying these methods and their numerous modifications designed for studying transcriptome profiles, searching for unidentified expressed loci, characterization of promoter regions, and high-throughput mapping of various genomic sites, such as hypo- and hypermethylated CpGs, and chromatin-binding and DNase I cleavage sites.

  26. Azhikina T., Gainetdinov I., Skvortsova Y., Sverdlov E. (2006). Methylation-free site patterns along a 1-Mb locus on Chr19 in cancerous and normal cells are similar. A new fast approach for analyzing unmethylated CCGG sites distribution. Mol. Genet. Genomics 275 (6), 615–22 [+]

    We describe a newly developed technique for rapid identification of positions of genomic DNA breaks, preexisting or introduced by specific digestion, in particular, by restriction endonucleases (RIDGES). We applied RIDGES in analyzing unmethylated CCGG sites distribution along a 1-Mb long genome region (D19S208-COX7A1 on chromosome 19) in cancerous and normal lung tissues. Both tissues were characterized by a profoundly uneven density of unmethylated sites along the fragment. Interestingly, the distribution of hypomethylated regions did not correlate with gene locations within the fragment, and one of the most hypomethylated areas contained practically no genes. We also demonstrated that the methylation pattern of a long genome DNA fragment was rather stable and practically unchanged in human lung cancer tissue as compared with its normal counterpart, in accordance with the suggestion (Ross et al. in Nat Genet 24:227-235, 2000) that cell lines of common origin have typically similar transcription profiles. An analogous suggestion might probably be made for global methylation patterns of genomic DNA.

  27. Azhikina T., Gvozdevsky N., Botvinnik A., Fushan A., Shemyakin I., Stepanshina V., Lipin M., Barry C. 3rd, Sverdlov E. (2006). A genome-wide sequence-independent comparative analysis of insertion-deletion polymorphisms in multiple Mycobacterium tuberculosis strains. Res. Microbiol. 157 (3), 282–90 [+]

    We applied an enhanced version of subtractive hybridization for comparative analyses of indel differences between genomes of several Mycobacterium tuberculosis strains widespread in Russian regions, and the H37Rv reference strain. A number of differences were detected and partially analyzed, thus demonstrating the practicality of the approach. A majority of the insertions found were shared by all Russian strains, except for strain 1540 that revealed the highest virulence in animal tests. This strain possesses a number of genes absent from other clinical strains. Two of the differential genes were found to encode putative membrane proteins and are presumed to affect mycobacterial interaction with the host cell, thus enhancing virulent properties of the isolate. The method used is of general application, and enables the elaboration of a catalogue of indel polymorphic genomic differences between closely related strains.

  28. Azhikina T.L., Sverdlov E.D. (2005). Study of tissue-specific CpG methylation of DNA in extended genomic loci. Biochemistry Mosc. 70 (5), 596–603 [+]

    Modern approaches for studies on genome functioning include investigation of its epigenetic regulation. Methylation of cytosines in CpG dinucleotides is an inherited epigenetic modification that is responsible for both functional activity of certain genomic loci and total chromosomal stability. This review describes the main approaches for studies on DNA methylation. Under consideration are site-specific approaches based on bisulfite sequencing and methyl-sensitive PCR, whole-genome approaches aimed at searching for new methylation hot spots, and also mapping of unmethylated CpG sites in extended genomic loci.

  29. Azhikina T., Kostina M., Skaptsova N., Potapov V., Berg D., Sverdlov E. (1996). Factors affecting the priming efficiency of short contiguous oligonucleotide strings in the primer walking strategy of DNA sequencing. DNA Seq. 6 (4), 211–6 [+]

    We use modified oligonucleotides with enhanced strength of complementary DNA binding for primer walking DNA sequencing with strings of short contiguous oligonucleotides as primers. Such an approach allows us to reduce the probability of primer failures due to unstable binding of oligos with templates. In this paper the factors affecting the priming efficiency of segmented primers (strings composed of several short oligonucleotides contiguously juxtaposed on the template) used for DNA sequencing were investigated. Modified oligonucleotides were used to discriminate the effects caused by intrinsic properties of the oligonucleotides and by template features. It was shown that the most crucial factor is the stability of the duplex formed by the template with the 3'-outermost oligonucleotide of a string. The data were obtained with a model M13 template and in the process of sequencing the region flanking a long terminal repeat of human endogenous retrovirus HERVK mapped on chromosome 19. The sequencing was done by primer walking with strings of contiguous modified hexanucleotides. The effects revealed should be taken into consideration when choosing oligonucleotide units of segmented primers and for construction of minimised libraries composed of short unit oligonucleotides.

  30. Azhikina T., Veselovskaya S., Myasnikov V., Potapov V., Ermolayeva O., Sverdlov E. (1993). Strings of contiguous modified pentanucleotides with increased DNA-binding affinity can be used for DNA sequencing by primer walking. Proc. Natl. Acad. Sci. U.S.A. 90 (24), 11460–2 [+]

    Modified oligonucleotides containing 5-methylcytidine and/or 2-aminoadenosine form tighter hybrids with DNA and are, therefore, more efficient primers for DNA sequencing as compared to their natural counterparts. Strings of contiguous modified pentanucleotides can be used for DNA sequencing by primer walking.