Avoidance of recognition sites of restriction-modification systems is a widespread but not universal anti-restriction strategy of prokaryotic viruses.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: Avoidance of recognition sites of restriction-modification systems is a widespread but not universal anti-restriction strategy of prokaryotic viruses.

Comparison of Methods of Detection of Exceptional Sequences in Prokaryotic Genomes.

Many proteins need recognition of specific DNA sequences for functioning. The number of recognition sites and their distribution along the DNA might be of biological importance. For example, the number of restriction sites is often reduced in prokaryotic and phage genomes to decrease the probability of DNA cleavage by restriction endonucleases. We call a sequence an exceptional one if its frequency in a genome significantly differs from one predicted by some mathematical model. An exceptional sequence could be either under- or over-represented, depending on its frequency in comparison with the predicted one. Exceptional sequences could be considered biologically meaningful, for example, as targets of DNA-binding proteins or as parts of abundant repetitive elements. Several methods to predict frequency of a short sequence in a genome, based on actual frequencies of certain its subsequences, are used. The most popular are methods based on Markov chain models. But any rigorous comparison of the methods has not previously been performed. We compared three methods for the prediction of short sequence frequencies: the maximum-order Markov chain model-based method, the method that uses geometric mean of extended Markovian estimates, and the method that utilizes frequencies of all subsequences including discontiguous ones. We applied them to restriction sites in complete genomes of 2500 prokaryotic species and demonstrated that the results depend greatly on the method used: lists of 5% of the most under-represented sites differed by up to 50%. The method designed by Burge and coauthors in 1992, which utilizes all subsequences of the sequence, showed a higher precision than the other two methods both on prokaryotic genomes and randomly generated sequences after computational imitation of selective pressure. We propose this method as the first choice for detection of exceptional sequences in prokaryotic genomes.

Role of Restriction-Modification Systems in Prokaryotic Evolution and Ecology.

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 foreign DNA. Comparative analysis of a large number of available bacterial genomes and methylomes clearly demonstrates that the role of R-M systems in bacteria is wider than only defense. R-M systems maintain heterogeneity of a bacterial population and are involved in adaptation of bacteria to change in their environmental conditions. R-M systems can be essential for host colonization by pathogenic bacteria. Phase variation and intragenomic recombinations are sources of the fast evolution of the specificity of R-M systems. This review focuses on the influence of R-M systems on evolution and ecology of prokaryotes.

[Search for protective antigens in Ixodes persulcatus (ixodidae) salivary gland extracts].

RT-PCR evaluation of the activity of eight Ixodes persulcatus salivary gland genes shows clear distinctions in their expression depending of the stage of tick feeding. Out of them, only Salp 10 and Salp 15 proteins may be regarded as candidates for protective antigens to develop anti-tick and anti-Borrelia vaccines. Firstly they play an important role in feeding a tick and modifying a host's immune response. Secondly, the increasing expression of the salp 10 and salp 10 genes begins at early tick feeding stages. Thirdly, the activity of these genes increases with the beginning of feeding by tens and hundreds times and keeps at this level until the third tick feeding stage is over.

[Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo].

Bone morphogenetic protein-2 (rhBMP-2) represents the osteoinductive protein factor which plays a dominant role in growth and regeneration of a bone tissue. In clinical practice the bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials are not produced. The fragment of the bmp2gene coding for a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on a basis of the E. coli BL21 (DE3). The rhBMP-2 production was about 25% of total cell protein. The biologically active dimeric form of rhBMP-2 was obtained by isolation and purification of protein from inclusion bodies with subsequent refolding. The rhBMP-2 sample with more than 80% of the dimeric form was obtained, which is able to interact with specific antibodies to BMP-2. Biological activity of the received rhBMP-2 samples was shown in the in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On model of the ectopic osteogenesis it was shown that received rhBMP-2 possesses biological activity in vivo, causing tissue calcification in the place of an injection. The protein activity in vivo depends on way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The offered method of rhBMP-2 isolation and purification results in increasing common protein yield as well as the maintenance of biologically active dimeric form in comparison with the analogues described in the literature.

[Changes in the expression of salivary gland genes in Ixodes persulcatus (Ixodidae) depending on the stage of tick feeding].

By using the guanidine-isothiocyanate test, the authors isolated a summary RNA preparation from Ixodes persulcatus salivary gland extracts. Activity products of the genes responsible for the expression of some salivary proteins were first identified using the RT-PCR. It has been shown that, firstly, I. persulcatus synthesizes at least 3 transcripts homologous to the respective salivary components of the related species I. scapularis, the translation product of which is likely to be immunodominant antigens; secondly, the number of each of these transcripts, as in I. scapularis, depends on the stage of tick feeding. The changes in the expression of each transcript are specific: monotonously increasing changes in Salp 17 and cyclic ones in Salp 16, and synthesis, only when the ticks are fully ingested, in Salp 25.

[Evaluation of the occurrence of borreliae in the ticks]. / Otsenka porazhennosti kleshchei borreliiami.

The paper shows it necessary to estimate the average number of borreliae in the infected ticks, by using the geometric mean of a sign rather than the arithmetic mean.

[A laboratory model of the parasitic system of Lyme disease]. / Laboratornaia model' parazitarnoi sistemy bolezni Laima.

A laboratory model of the parasitic system of Lyme borreliosis (LB) with Ixodes persulcatus and Borrelia burgdorferi s.s. underwent 5 epizootic cycles (Cycle I transition of Borreliae by the scheme a tick-->a mouse-->a tick corresponds to the seasonal cycle). A group of 5-6 mice was inoculated with the suspension of infected nymphs by the intracutaneously route and 35-45 days later intact tick larvae or nymphs were fed on them. In the first three cycles, 40-100% of mice in a group (a total of 33 of 59) were susceptible, but 3 of 11 groups of mice were not inoculated. 20-50% of larvae and 75-92% of nymphs were susceptible on the infected mice. The mean geometric abundance of borreliae was 2.8-6.3 cells per 100 fields of vision in the nymphs infected in the larval phase and 16.1-29.5 cells in the image phase. In cycle IV, 2 of 6 groups of mice were unsusceptible to borreliae and 5 of the 17 mice from 4 groups got infected. One mouse died from Lyme borreliosis when larvae were fed on it. The nymphs remolted from underfed larvae were infected in 62.2% of cases, the mean geometric abundance in these nymphs was 5.7% per 100 fields of vision. A total of 3.2-14.7% of the ticks fed on the remaining 4 mice were infected and the abundance of boreliae was 1-2 per 100 fields of vision. In cycle V, one of 12 mice got infected, the infection rate in nymphs was 9.1%, the abundance of borreliae was 1 per 100 fields of vision. Thus, as the number of epizootic cycles increases, the proportion of infected mice, that of ticks infected on these mice on feeding, and the abundance of borreliae in the infected ticks decrease. Moreover, there was a reduction in the length of borreliae reduced from 17-18 mm to 10-11 mm and in the duration of multiplication and preservation of borreliae in the infected ticks and there was concurrently an increase in the duration of development of an infectious process in mice. It is suggested that the population of borreliae consists of 2 morphophysiological groups or more. During cultivation of the laboratory model of the parasitic system of Lyme borreliosis, a group of borrelia that are short and slightly pathogenic for mice had got many points in its favour. A five-fold procedure of artificial election of precisely defining this group set the model on the verge of disappearance. The capacity of the populations of borreliae to alter their properties rapidly and fundamentally suggests that there might be violent annual fluctuations in human morbidity.

[An experimental study of the capacity of the rat mite Ornithonyssus bacoti (Hirst, 1913) to ingest, maintain and transmit Borrelia]. / Eksperimental'noe izuchenie sposobnosti krysinogo kleshcha Ornithonyssus bacoti (Hirst, 1913) vosprinimat', sokhraniat' i peredavat' borrelii.

For the first time a possibility of the gamasina mites' O. bacoti participation in Lyme disease spirochetes' circulation has been demonstrated. It has been experimentally shown that Borrelia burgdorferi s.l. are received by O. bacoti, survive in them for at least 21 days and are transmitted to white mice through mites' bites. Mice's infestation has occurred in 23% of cases. It is suggested that other bloodsucking gamasina mites inhabiting the Lyme borreliosis reservoir rodents nests may be capable of participating in borrelia circulation in the Lyme disease endemic areas.

[Propagation of the pathogen of Lyme disease Borrelia burgdorferi in the ticks Ixodes persulcatus]. / Razmnozhenie vozbuditelei bolezni Laima Borrelia burgdorferi v kleshchakh Ixodes persulcatus.

An increase of the number of Borrelia burgdorferi s. l. in nymphs was observed 3-4 months later the moulting and during the following 4-5 months. The percent of infected ticks remained unchanged. The reproduction of spirochete was going not only at a room temperature, but also at a lower temperature, 3-5 degrees (imitation of hibernation conditions). An increase of the spirochete density was especially well marked, when the ticks were maintained under fluctuated daily conditions. We have found out the difference in the transphase transmission. Almost all larvae and nymphs infected by a bloodsucking retained spirochetes after moulting (the first transphase transmission). When the nymphs infected at larval stage were fed on an uninfected mouse, only 30% of imago ticks retained the spirochetes (the second transphase transmission).

[The reaction of taiga ticks to an attractant. II. The rates of removing ticks from a population]. / Reaktsiia taezhnykh kleshchei na attraktant. Soobshchenie II. Tempy iz''iatiia kleshchei iz populiatsii.

Catching ticks on the attractant-moistened napkins and on the flag (control) under field conditions during 5 days has demonstrated that a population of taiga ticks on the treated areas can be killed within 1-2 weeks when attractive-acaricidal granules are applied.

[The activity of cyfluthrin and cypermethrin-based preparations against the vector of the causative agent of tick-borne relapsing fever]. / Aktivnost' tsiflutrina i preparatov na osnove tsipermetrina po otnosheniiu k perenoschiku vozbuditelia kleshchevogo vozvratnogo tifa.

Laboratory tests have established a high activity and long-term persistence of some cipermethrin isomers (fendone, fastac) and cifluthrin (zolfac) against the tick Ornithodoros papillipes, a carrier of tick-borne relapsing fever. These indices are considerably superior to those of an isomer mixture of cipermethrin (cimbush, ripkord). This can be applied to deltamethrin and cigalothrin. The findings suggest that these agents are highly promising in controlling the carrier of tick-borne relapsing fever along with a significant reduction in the ecological pesticidal load in the focus.

[The rate of infection of the taiga tick with Borrelia in the western Sayan]. / Zarazhennost' taezhnogo kleshcha borreliiami v Zapadnom Saiane.

The paper provides microscopic findings of taiga ticks collected for the causative agent of Lyme's disease in May to July 1993 in the West Sayan . A total of 1,016 specimens were examined, among them 124 (12.2%) were found to have Borreliae. The areas were demonstrated to vary greatly with their epidemiological risk. The number of ticks and hence the epidemiological risk were the most in the low-mountain and chern taiga areas.

[The sensitivity of the tick Ornithodoros papillipes Bir. to Icon]. / Chuvstvitel'nost' kleshcha Ornithodoros papillipes Bir. k aikonu.

Laboratory trials have demonstrated a high and stable acaricidal activity of aikon towards O. papillipes ticks. The minimal dose (on filter paper) of aikon providing a 100% death of the ticks at all developmental phases, effective for at least 9-12 months, was 30 mg/m2. In exposure to this dose the ticks died within 1-7 days; if impregnated filter paper was stored for a long time, within 15-30 days. The lower the dose was, the slower the effect and lesser the total death rate of the ticks were.

[Changes in the village foci of tick-borne relapsing fever in Uzbekistan over a 10-year period]. / Ob izmeneniiakh poselkovykh ochagov kleshchevogo vozvratnogo tifa v Uzbekistane za desiatiletnii period.

The paper presents the results of repeated investigations in the tick relapsing fever nidus (TRF) conducted after a ten-year interval in Gava settlement situated in the foothill area of the Namangan Province of Uzbekistan. A considerable deterioration in the habitat conditions of the vector, Ornithodoros papillipes Bir., due to the intensified anthropogenic effect, and decrease in its number have been noted. The distribution range of the tick (percent of infested farmsteads) in this case did not reduce. The infection rate of the vector's population with the tick relapsing fever agent decreased that points indirectly to a higher mortality of infected ticks under unfavourable conditions. Wavy course of morbidity with TRF during the period between investigations shows that changes that took place in the nidus were not uniform in their character and may have resulted to some extent from natural cycles. A similar character of anthropogenic effect on the populations of ticks in the settlement natural nidi suggests that the above changes are common to the nidi of this infection.

[Tick-borne relapsing fever morbidity in the western Pamirs]. / Zabolevaemost' kleshchevym vozvratnym tifom na Zapadnom Pamire.

Morbidity with tick-borne recurrent fever in the western Pamirs is compared with that of the 1930s. It reduced considerably though the disease is widespread in the Pandj valley. The majority of patients are earlier resistant local inhabitants. This may be accounted for by vector number decrease to the level causing loss of immunity in the large part of the population. Unlike even distribution of recurrent fever in the western Pamirs in the 1930s, nowadays most cases are detected in the lower parts of the Pandj valley and there are no infection foci in the upper parts, which might be connected with gradual increase in the portion of immune population up the Pandj river and with diagnosis inadequacy. The incidence of recurrent fever in the Pamirs is considerably higher than in other parts of Tajikistan.

[The current status of foci of tick-borne relapsing fever in the western Pamirs]. / Sovremennoe sostoianie ochagov kleshchevogo vozvratnogo tifa na Zapadnom Pamire.

For the first time in the last 20-50 years foci of tick-borne relapsing fever have been surveyed in the western Pamirs. Settlements in the Piandzha valley were shown to be (as in the 1930s and 1960s) affected by Ornithodoros papillipes Bir., the carrier of the infection. Though the number of ticks is still rather high, it is nowadays considerably reduced. The findings suggest a tendency towards a decrease in the infestation of the carrier population with the agent of the disease. The traditional type of construction (especially widespread in far-off regions), when houses are built in such a way that living rooms are joined with stables inhabited by ticks, ensures favourable conditions for the existence of tick-borne relapsing fever foci and the contact of people with the agent of the infection. This demonstrates high epidemiological danger of the infection foci in the western Pamirs.

[The effect of the causative agent of tick-borne relapsing fever on the respiration of the vector Ornithodoros papillipes (Birula, 1895) (Ixodoidea, Argasidae)]. / Vliianie vozbuditelia kleshchevogo vozvratnogo tifa na dykhanie perenoschika Ornithodoros papillipes (Birula, 1895) (Ixodoidea, Argasidae).

A relatively low level of normal respiratory metabolism in O. papillipes has been observed as compared to most other invertebrates and even ixodid ticks. It is connected with biological peculiarities and habitat of this species. Changes in respiratory metabolism under the influence of Borrelia have been revealed. These changes vary at different stages of tick life cycle. In long-fasting infected individuals, respiration is further inhibited, and in nourished ones, is increased as compared to noninfected ticks.

[Effect of Borrelia--the causative agent of tick-borne relapsing fever--on the vector]. / Vliianie borrelii--vozbuditelei kleshchevogo vozvratnogo tifa--na perenoschika.

Direct or indirect evidence of pathogenic effect of Borrelia sogdiana, a tick-borne recurrent fever causative agent, on Ornithodoros papillipes is obtained, pointing to a parasitic character of their relations. Differences between infected and uninfected ticks are revealed both under normal life conditions (lower survival, more intensive process of moult), as well as under the effect of poison. Higher sensitivity of infected ticks to DDT was established experimentally; HCCH effect was determined indirectly by counting ticks after treatment. Authors suggest that the effect of causative agent on ticks' viability is more expressed under the conditions close to emergency.