Restriction–modification (R-M) systems are able to methylate or cleave DNA depending on methylation status of their recognition site. It allows them to protect bacterial cells from invasion by...

Restriction-modification (R-M) systems protect bacteria and archaea from attacks by bacteriophages and archaeal viruses. An R-M system specifically recognizes short sites in foreign DNA and cleaves it, while such sites in the host DNA are protected by methylation. Prokaryotic viruses have developed a number of strategies to overcome this host defense. The simplest anti-restriction strategy is the elimination of recognition sites in the viral genome: no sites, no DNA cleavage. Even a decrease of the number of recognition sites can help a virus to overcome this type of host defense. Recognition site avoidance has been a known anti-restriction strategy of prokaryotic viruses for decades. However, recognition site avoidance has not been systematically studied with the currently available sequence data. We analyzed the complete genomes of almost 4000 prokaryotic viruses with known host species and more than 17,000 restriction endonucleases with known specificities in terms of recognition site avoidance. We observed considerable limitations of recognition site avoidance as an anti-restriction strategy. Namely, the avoidance of recognition sites is specific for dsDNA and ssDNA prokaryotic viruses. Avoidance is much more pronounced in the genomes of non-temperate bacteriophages than in the genomes of temperate ones. Avoidance is not observed for the sites of Type I and Type IIG systems and is very rarely observed for the sites of Type III systems. The vast majority of avoidance cases concern recognition sites of orthodox Type II restriction-modification systems. Even under these constraints, complete or almost complete elimination of sites is observed for approximately one-tenth of viral genomes and a significant under-representation for approximately one-fourth of them. Avoidance of recognition sites of restriction-modification systems is a widespread but not universal anti-restriction strategy of prokaryotic viruses.

Avoidance of palindromic recognition sites of Type II restriction-modification (R-M) systems was shown for many R-M systems in dozens of prokaryotic genomes. However the phenomenon has not been investigated systematically for all presently available genomes and annotated R-M systems. We have studied all known recognition sites in thousands of prokaryotic genomes and found factors that influence their avoidance. Only Type II R-M systems consisting of independently acting endonuclease and methyltransferase (called ‘orthodox’ here) cause avoidance of their sites, both palindromic and asymmetric, in corresponding prokaryotic genomes; the avoidance takes place for ~ 50 % of 1774 studied cases. It is known that prokaryotes can acquire and lose R-M systems. Thus it is possible to talk about the lifespan of an R-M system in a genome. We have shown that the recognition site avoidance correlates with the lifespan of R-M systems. The sites of orthodox R-M systems that are encoded in host genomes for a long time are avoided more often (up to 100 % in certain cohorts) than the sites of recently acquired ones. We also found cases of site avoidance in absence of the corresponding R-M systems in the genome. An analysis of closely related bacteria shows that such avoidance can be a trace of lost R-M systems. Sites of Type I, IIС/G, IIM, III, and IV R-M systems are not avoided in vast majority of cases. The avoidance of orthodox Type II R-M system recognition sites in prokaryotic genomes is a widespread phenomenon. Presence of an R-M system without an underrepresentation of its site may indicate that the R-M system was acquired recently. At the same time, a significant underrepresentation of a site may be a sign of presence of the corresponding R-M system in this organism or in its ancestors for a long time. The drastic difference between site avoidance for orthodox Type II R-M systems and R-M systems of other types can be explained by a higher rate of specificity changes or a less self-toxicity of the latter.

Standard Affymetrix technology evaluates gene expression by measuring the intensity of mRNA hybridization with a panel of the 25-mer oligonucleotide probes, and summarizing the probe signal intensities by a robust average method. However, in many cases, signal intensity of the probe does not correlate with gene expression. This could be due to the hybridization of the probe to a transcript of another gene, mapping of the probe to an intron, alternative splicing, single nucleotide polymorphisms and other reasons. We have developed a database, PLANdbAffy (available at http://affymetrix2.bioinf.fbb.msu.ru), that contains the results of the alignment of probe sequences from five Affymetrix expression microarrays to the human genome. We have determined the probes matching the transcript-coding regions in the correct orientation. For each such probe alignment region, we determined the mRNA and EST sequences that contain the probe sequence. In the textual part of the database interface we summarize the data on the sequences that cover the probe alignment region and SNPs that are located inside it. The graphical part of our database interface is implemented as custom tracks to the UCSC genome browser that allows one to utilize all the data that are offered by UCSC browser.

E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference “Tick-E-Vac” vaccine.

Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding MTase, an RE of Type II R-M system is highly toxic for the cell. Genes of the R-M system are linked in the genome in the vast majority of annotated cases. There are only a few reported cases in which the genes of MTase and RE from one R-M system are not linked. Nevertheless, a few hundreds solitary RE genes are present in the Restriction Enzyme Database (http://rebase.neb.com) annotations. Using the comparative genomic approach, we analysed 272 solitary RE genes. For 57 solitary RE genes we predicted corresponding MTase genes located distantly in a genome. Of the 272 solitary RE genes, 99 are likely to be fragments of RE genes. Various explanations for the existence of the remaining 116 solitary RE genes are also discussed.