Use of Genetically Encoded Fluorescent Aptamers for Visualization of Mycobacterium tuberculosis Small RNA MTS1338 in Infected Macrophages.

The possibility to visualize small bacterial RNAs inside macrophages infected with Mycobacterium tuberculosis was demonstrated for the first time. A macrophage cell line was infected with the M. tuberculosis strain expressing small noncoding mycobacterial RNA MTS1338 fused with an RNA aptamer, which could bind a fluorophore and trigger its fluorescence. As a result, treatment of the infected macrophages with the DFHBI-1T fluorophore allowed fluorescence-based detection of the aptamer-labeled MTS1338 both in mycobacteria and in the host cell cytoplasm. This system can significantly aid in revealing the role of small M. tuberculosis RNAs in the pathogenesis of tuberculosis through identification of their secretion routes and eukaryotic targets and elucidation of the associated molecular pathways.

Role of Small Noncoding RNAs in Bacterial Metabolism.

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.

MIRA analysis of RARß2 gene methylation in DNA circulating in the blood in lung cancer.

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 RARß2 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.

[Expression of Mycobacterium tuberculosis small RNAs in mice models of tuberculosis].

Identification of mechanisms that contribute to bacterial pathogens survival in the infected organism is a powerful approach to influence the pathogenic bacteria. Recently it was established that bacteria use small RNAs to regulate their metabolism. We studied the expression level of the three most highly expressed M. tuberculosis small RNAs MTS0997, MTS 1338 and MTS2823 at different stages of infection in. mice with different genetic resistance to tuberculosis. The maximum expression level of these small RNAs was observed at the early stages of infection.

Mycobacterium tuberculosis Transcriptome Profiling in Mice with Genetically Different Susceptibility to Tuberculosis.

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.

[Adaptive changes of Mycobacterium tuberculosis gene expression during the infectious process].

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.

[Identified small RNAs of Mycobacterium avium].

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.

[Transcriptome analysis of bacterial pathogens in vivo: problems and solutions].

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.

Adaptive Changes in Mycobacterium avium Gene Expression Profile Following Infection of Genetically Susceptible and Resistant Mice.

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.

Studies on functional role of DNA methylation within the FXYD5-COX7A1 region of human chromosome 19.

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.

[Genome similarity of Baikal omul and sig].

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.

[Differential expression of DTSsa4 Tc1-like transposons in closely related populations of Baikal ciscoes].

Two representatives of Baikal ciscoes - lake cisco and omul - diverged from a common ancestor as recently as 10-20 thousand years ago. We have found an increasing expression level of DTSsa4 Tc1-like DNA transposons in cisco and omul brains. The mapping of the sequences of these transposons from Salmo salar and Danio rerio genomes has shown that in some cases, these transposons are located in the 5' and 3' regions, as well as in the promoter regions of various genes. Probably, Tc1-like transposons affect the activity of neighboring genes, providing the adaptive divergence of the cisco population.

Use of short representative sequences for structural and functional genomic studies.

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.

[Deletion-insertion differences between the genomes of Mycobacterium tuberculosis highly virulent strain HN878 and less virulent strain CDC1551].

The modfied version of the method of subtracting hybridization for full-genome comparison of M. tuberculosis strain HN878, capable of inducing a nonpulmonary form of tuberculosis, with strain CDC1551 causing tuberculosis--with classical pulmonary symptoms. The clone library of differential fragments, responsible for differences between genome HN878 and genome CDC1551, was created. As the result of the structural analysis carried out in this study, the set of differential fragments was divided into. three main groups: new places of the integration of transposon IS6110; fragments resulting from the transformations of other repeating sequences of the genome; long unique nucleotide sequences, absent in genome CDC1551. Genome transformations may be a highly important factor of the modulation of the phenotypical properties of the pathogen, including those which jointly determined its virulence, and also served as valuable molecular genetic markers for diagnostic purposes.

Study of tissue-specific CpG methylation of DNA in extended genomic loci.

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.

Full-sized HERV-K (HML-2) human endogenous retroviral LTR sequences on human chromosome 21: map locations and evolutionary history.

One of the evolutionary mechanisms for acquisition of novel functional sequences can be domestication of exogenous retroviruses that have been integrated into the germ line. The whole genome mapping of such elements in various species could reveal differences in positions of the retroviral integration and suggest possible roles of these differences in speciation. Here, we describe the number, locations and sequence features of the human endogenous retrovirus HERV-K (HML-2) long terminal repeat (LTR) sequences on human chromosome 21. We show that their distribution along the chromosome is not only non-random but also roughly correlated with the gene density. Amplification of orthologous LTR sites from a number of primate genomes produced patterns of presence and absence for each LTR sequence and allowed determination of the phylogenetic ages and evolutionary order of appearance of individual LTRs. The identity level and phylogenetic age of the LTRs did not correlate with their map locations. Thus, despite the non-random distribution of LTRs, they have apparently been inserted randomly into the chromosome relative to each other. As evidenced in previous studies of chromosomes 19 and 22, this is a characteristic of HERV-K integration.

[Identification and mapping on human chromosome 19 of chromosome-specific repeated element preferentially binding with nuclear matrix]. / Identifikatsiia i kartirovanie na khromosome 19 cheloveka khromosomspetsificheskogo povtoriaiushchegosia élementa, predpochtitel'no sviazyvaiushchegosia s iadernym matriksom

From a library of sequences binding preferentially to nuclear matrix (matrix attachment regions, MARs), a fragment of about 300 bp in length (CEA (carcinoembryonic antigen)-MAR) was isolated and characterized. The CEA-MAR sequence was found in more than ten loci of chromosome 19 containing elements similar to genes of the CEA family. No sequences of this group were found on other human chromosomes. Two CEA-MAR-containing loci were sequenced, and sequences for another seven loci were found in GenBank. A comparative analysis of CEA-MARs and the flanking sequences is reported. Based on the sequence of the CEA-containing chromosome 19 loci, a hypothetical model of the domain structure of a 2-Mb chromosome region was constructed and the mutual arrangement of CEA-MARs and genes of CEA family was elucidated. The CEA-MARs were located 5-20 kb downstream of the CEA genes. These results suggest that the duplication unit of the CEA family may coincide with chromatin domains containing these genes.

[Structural characteristics of four long terminal repeats (LTR) of human endogenous retroviruses and features of their integration sites]. / Strukturnye kharakteristiki chetyrekh dlinnykh kontsevykh povtorov (LTR) endogennykh chelovecheskikh virusov i osobennosti uchastkov ikh integratsii.

Four LTR-containing regions of human chromosome 19 were sequenced by the primer walking technique using strings of short oligonucleotides tightly bound to the template. A comparative and evolutionary analysis of sequences homologous to human endogenous retroviruses (HERV) was performed, and the prototypes of the LTRs were determined. Analysis of the chromosome 19 sequences adjacent to LTR revealed that LTRs of HERV-K share a common location with other retroposons.

Determination of the patterns of hexanucleotide distribution along DNA by electron microscopy.

Hexanucleotides containing modified bases (5-methylcytosine and 2-aminoadenine instead of cytosine and adenine) with increased capacities to bind complementary DNA sequences were used to map the distribution of their complementary sequences in a DNA target using electron microscopy. The method used hexamers to initiate DNA polymerase directed DNA synthesis at complementary sequences along a template. DNA synthesis was limited to about 200 residues by using a low concentration of deoxynucleotide precursors. During DNA synthesis a biotin ligand was incorporated to facilitate the subsequent binding of an electron-dense label (streptavidin-labeled colloidal gold particles) into newly synthesized DNA chains. The method can be implemented with commercially available products. The results demonstrate that the approach can be used to compare primary structural features of DNA fragments. The principles of the method can be adapted to a variety of single molecule detection methods such as electron, scanning tunneling, or atomic force microscopies.