Development of Methodology for Analyzing RNA Structure and Its Application in Molecular Biology and Virology.

The review covers three independent blocks of research. The first one is discovery, isolation, and investigation of snake venom RNases and their use in studying RNA macrostructure. It has been established that snake venom RNases are not specific to the primary RNA structure but rather to the RNA helical conformation (double, single, or hybrid helix). Snake venom RNases hydrolyze RNA to short oligomers with the 5'-terminal phosphate. Analysis of the kinetics and products of tRNA hydrolysis exemplifies the use of snake venom RNases for deciphering RNA macrostructure. The second block is devoted to the principle formulated by the author for analyzing the primary structure of nucleic acids and describes the method of direct RNA sequencing that has been developed with author's participation. The third block describes the results of genotyping and etiologic control of epidemic influenza A viruses circulating in the Soviet Union in 1968 to 1992. The method for comparative analysis of genome sequences of viral isolates has made it possible to detect and characterize epidemic influenza virus strains that had emerged in the circulation as a result of reactivation of inactivated vaccines.

[Selection of initiation replication mutants of IncP-9 plasmid pBS267].

A minireplicon containing the rep gene and oriV site of the gamma subgroup of the IncP-9 caprolactam pBS267 biodegradation plasmid was cloned for the first time. It was established that a minimized variant of pBS267 plasmid cannot be sustained in E. coli and is inherited in an unstable way in bacteria Pseudomonas. Using in vitro mutagenesis, mutant variants of the minireplicon were produced, characterized by an increased number of copies in cells, the ability to replicate in E. coli, and relatively stable inheritance in P. putida cells. The obtained constructs are the basis for a study of the replication mechanisms of IncP-9 group plasmids, as well as use as vectors for molecular cloning in a wide range of gram-negative bacteria.

[Inheritance of biodegradation plasmids in the cells of homo- and heterologous hosts].

A systematic analysis of the inheritance of D plasmids of the IncP-9 group (alpha-, beta-, gamma-, delta-, epsilon-, zeta-, eta-, theta-subgroups), IncP-7, as well as of those of undefined systematic affiliation in the cells of homologous (Pseudomonas putida) and heterologous (Escherichia coli) hosts was performed for the first time. For this purpose, mini-Tn5 transposons determining resistance to kanamycin (or streptomycin) were introduced into all the D plasmids under study. It has been established that all IncP-9 plasmids can be transmitted to the cells of a heterologous host E. coli (with the exception of plasmid pSVS15 from theta-subgroup). IncP-7 plasmids and those of undefined systematic affiliation do not possess this property and can be transmitted and stably inherited only in P. putida. The distinctive feature of most IncP-9 plasmids (alpha-, beta-, gamma-, delta-, epsilon-, and zeta-subgroups) is strict dependence of their inheritance on the temperature factor. At 37 degrees C, the plasmids of delta-, zeta-, and theta-subgroups are unstable in P. putida cells, while in E. coli nearly all plasmids of this systematic group are unstable. The exceptions are the plasmids of eta- and gamma-subgroups. Inheritance of these plasmids does not depend on temperature. At 28 degrees C and 37 degrees C, the eta plasmid is not maintained stably (inheritance stability is 2%), while the gamma plasmid has almost 100% stability under the same conditions.

[Characteristics of natural strains of naphthalene-utilizing bacteria of the genus Pseudomonas].

Sixty-three strains of bacteria capable of utilizing naphthalene as the sole source of carbon and energy were isolated from 137 samples of soil taken in different sites in Belarus. All isolated bacteria contained extrachromosomal genetic elements of 45 to 150 kb in length. It was found that bacteria of 31 strains contained the IncP-9 incompatibility group plasmids, bacteria of one strain carried a plasmid containing replicons IncP-9 and IncP-7, and bacteria of 31 strains contained unidentified plasmids. Primary identification showed that the hosts of plasmids of naphthalene biodegradation are fluorescent bacteria of the genus Pseudomonas (P. putida and P. aeruginosa; a total of 47 strains) and unidentified nonfluorescent microorganisms (a total of 16 strains). In addition to the ability to utilize naphthalene, some strains exhibited the ability to stimulate the growth and development of the root system of Secale cereale.

[Functional abilities of the teenager's body based on body mass index]. / Funktsional'nye vozmozhnosti organizma podrostkov v zavisimosti ot indeksa massy tela.

The nutrition status was investigated according to the body weight index (BWI) in teenagers studying at different educational facilities. 77.1% of male teenagers and 70% of female teenagers from among all examined subjects (1456 persons) were assigned to the group with normal BWI ranging from 18.5 to 25.0 kg/m2. Male teenagers with a lower BWI (below 18.5 kg/m20) were more often encountered in boarding schools and specialized technical-and-professional schools; as for female teenagers, subjects with a lower BWI were more often encountered in general education schools and among first-year students of high schools (24 and 43.75%, respectively). An insignificant number of teenagers with an excessive body weight was detected in all educational facilities. Finally, it was concluded that teenagers with normal BWI have the best indices of functional abilities.

[Conjugation mapping of Pseudomonas mendocina bacteria]. / Kon''iugatsionnoe kartirovanie bakterii Pseudomonas mendocina.

Donor strains of the Hfr type were isolated using plasmid pRK2013 with transposons Tn10 and Tn5 as a chromosome-mobilizing factor. The isolated strains were shown to promote transfer of donor chromosome from different origins in different directions during isogenic matings of Pseudomonas mendocina bacteria. The created collection of donors and polyauxotrophic recipient bacteria permitted mapping 26 genetic determinants on the bacterial chromosome and identifying the genome of these microorganisms as a circular DNA molecule.

[Construction of a genetic map of Pseudomonas mendocina bacteria]. / Postroenie geneticheskoi karty bakterii Pseudomonas mendocina.

Based on the results of matings with interrupted conjugation and analysis of marker joint inheritance frequencies, distances between 26 genetic determinants were estimated and a genetic map of Pseudomonas mendocina bacteria was constructed.

[Characteristics of body composition of the students of boarding schools]. / Kharakteristika komponentov massy tela uchashchikhsia obshcheobrazovatel'nykh shkol-internatov.

The article describes the findings on the peculiarities of body composition of pupils of boarding high schools in Vitebsk region in the 1990s. The age- and sex-dependent dynamics of body components was shown in children aged 8-15 years in absolute values and as percentage in relation to total the body mass.

[Constructing of system of genetic analysis in Pseudomonas mendocina]. / Sozdanie sistemy geneticheskogo analiza bakterii Pseudomonas mendocina

A Tn10-containing variant of the pRK2013 plasmid, pRK2013-7, was used in the genetic analysis of Pseudomonas mendocina as chromosome-mobilizing inheritable factor that is able to integrate into the bacterial chromosome and transfer genetic markers with a frequency ranging from 3.2 x 10(-7) to 3.5 x 10(-3). The results of interrupted matings allowed localization of 10 genetic markers. This system of genetic analysis is suitable for P. mendocina mapping.

[Results of experimental radiobiological studies made within the 10-kilometer zone of the Chernobyl AES accident]. / Itogi éksperimental'nykh radiobiologicheskikh issledovanii v 10-kilometrovoi zone avarii na ChAES.

A study was made of the effect of high radioactive contamination on the animal organism (C57BL/6 mice) and HeLa cell culture within the ten-kilometer zone of the Chernobyl A.P.S. accident. The total radiation dose, as calculated by a gamma-component, was 0.09 to 2 Gy. A long-term exposure of mice within the zone (cumulative dose of 1.8 to 2 Gy) caused a significant decrease in bone marrow stem potencies and changes in the brain vascular system; subsequent acute exposure of animals increased interferon titres in the serum to a much greater extent than a single acute exposure did. As to HeLa cells, irradiation there of with doses of 0.09 to 0.4 Gy during 15-20 postirradiation generations caused a decrease in the proliferative activity, an emergence of cells with micronuclei and of giant cells, and remote cell death.

[The characteristics of influenza A/H1N1 viruses related to A/PR/8/34 isolated in the Mongolian People's Republic]. / Kharakteristika virusov grippa A/H1N1, rodstvennykh A/PR/8/34, izolirovannykh v MNR.

Investigations of the antigenic structure and genome of influenza A (H1N1) viruses isolated in the Mongolian People's Republic in 1982, 1983, 1986 and 1987 from children with acute respiratory diseases using monoclonal antibodies and nucleotide sequencing revealed 4 strains identical to the prototype strain A/PR/8/34 (H1N1), variant M. Sinai, and 8 strains closely similar to the epidemic strain A/USSR/90/77 (H1N1) in the antigenic structure and A/Leningrad/54 (H1N1) in the primary structure of HA.

[Hybridization of DNA with oligonucleotides immobilized in a gel: a convenient method for recording single base replacements]. / Gibridizatsiia DNK s oligonukleotidami, immobilizovannymi v gele: udobnyi metod registratsii odinochnykh zamen osnovanii.

In an attempt to develop a reliable system for DNA sequence analysis with multiple hybridization probes, oligonucleotides down to 8 bases long were covalently immobilized in a thin layer of polyacrylamide gel fixed on a glass plate. It was shown possible to detect single base changes in DNA by hybridization of the immobilized oligonucleotides with radioactively and fluorescently labeled DNA fragments. Moreover, it was found that dissociation temperatures of differently GC-rich duplexes could be equalized by appropriate choice of immobilized oligonucleotides concentrations. A model accounting for this phenomenon is presented. In order to make the system more compact, a rectangular matrix of 200 mm dots of immobilized oligonucleotides ("hybridization chip") was designed which offered the sensitivity of 20 attomoles per dot for fluorescent DNA fragment. The applications and perspectives of the approach are discussed.

A method for DNA sequencing by hybridization with oligonucleotide matrix.

A new technique of DNA sequencing by hybridization with oligonucleotide matrix (SHOM) which could also be applied for DNA mapping and fingerprinting, mutant diagnostics, etc., has been tested in model experiments. A dot matrix was prepared which contained 9 overlapping octanucleotides (8-mers) complementary to a common 17-mer. Each of the 8-mers was immobilized as individual dot in thin layer of polyacrylamide gel fixed on a glass plate. The matrix was hybridized with the 32P-labeled 17-mer and three other 17-mers differing from the first one by a single base change. The hybridization enabled us to distinguish perfect duplexes from those containing mismatches in 32 out of 35 cases. These results are discussed with respect to the applicability of the approach for sequencing. It was shown that hybridization of DNA with an immobilized 8-mer in the presence of a labeled 5-mer led to the formation of a stable duplex with the 5-mer only if the 5- and the 8-mers were in continuous stacking making a perfect nicked duplex 13 (5+8) base pairs long. These experiments and computer simulations suggest that continuous stacking hybridization may increase the efficiency of sequencing so that random or natural coding DNA fragments about 1000 bases long could be sequenced in more than 97% of cases. Miniaturized matrices or sequencing chips were designed, where oligonucleotides were immobilized within 100 x 100 micron dots disposed at 100 micron intervals. Hybridization of fluorescently labeled DNA fragments with microchips may simplify sequencing and ensure sensitivity of at least 10 attomoles per dot. The perspectives and limitations of SHOM are discussed.

[Modern variations of human influenza group A viruses at the molecular level]. / Sovremennoe raznoobrazie virusov grippa A cheloveka na molekuliarnom urovne.

The authors own results on the variety of the genomic primary structures in human influenza A viruses participating in the epidemic process, including the atypical viruses. The comparative studies revealed new trends in the HA gene antigenic drift on the late stages and the PB1 gene shift. Modifications occurring in the primary structure of the influenza A viruses native genomes during laboratory treatment (adaptation to new hosts, vaccine preparation, egg passaging) have been analyzed. Sequencing of several types of "antigenic anachronisms" revealed the direct links between some of such viruses and the anthropogenic pollution of the biosphere by vaccine strains. Modifications in the HA genes of influenza A viruses during the persistent infection have also been studied.

[Primary structure of the hemagglutinin gene in strains of the influenza virus A/Leningrad/624/86 and A/Leningrad/621/86 serologic subtype H1N1]. / Pervichnaia struktura gena gemaggliutinina shtammov virusa grippa A/Leningrad/624/86 i A/Leningrad/621/86 seropodtipa H1N1.

A molecular analysis was made of genomes of influenza A (H1N1) virus strains, the causative agents of an epidemic in Leningrad, 1986. The primary structure of hemagglutinin gene of two of these strains, A/Leningrad/624/86 and A/Leningrad/621/86, was established, as well as partial primary structure of PB1 gene of certain current strains of the A (H1N1) subtype. A hypothesis of a "shift" of PB1 gene in 1950-1957 is suggested.