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A general scheme for the synthesis of ribonucleotide monomers containing alkoxymethyl group in 2'-O-position for the solid-phase phosphotriester oligonucleotide synthesis using O-nucleophilic intramolecular catalysis has been developed. In particular, the monomers containing 2'-O-modifying 2-azidoethoxymethyl, propargyloxymethyl, or 3,4-cyclocarbonatebutoxymethyl groups has been prepared.

An efficient synthetic method for monomer ribonucleotide synthons containing 2'-O-methoxymethyl and 2'O-(p-nitrobenzyloxy)methyl groups used for oligonucleotide phosphotriester method with O-nucleophilic intramolecular catalysis at the stage of formation of internucleotide bond is developed. It is shown that synthons containing protecting 2'-O-(p-nitrobenzyloxy)methyl group may be used for automatic synthesis of phosphotriester oligoribonucleotides with high yields and synthons containing methoxymethyl group--to get 2'-O-modified oligonucleotides.

Conjugate the fluorescent dye 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indatsen-8-propionic acid (BODIPY) and N2,N4,N6-trimethylmelamine was obtained. It was shown that this compound in the presence of formaldehyde generates covalent cross-links of DNA strands in vitro.

In order to improve physicochemical and biological properties of natural oligonucleotides in particular increasing their affinity for nucleic acids, the selectivity of action and biological sustainability, several types of DNA mimics were designed. The survey collected data on the synthesis and properties of the DNA mimics - peptide-nucleic acids analogues, which are derivatives of pyrrolidine and hydroxyproline. We examine some physicochemical and biological properties of negatively charged mimics of this type, containing phosphonate residues, and possessing a high affinity for DNA and RNA, selective binding with nucleic acids and stability in various biological systems. Examples of the use of these mimics as tools for molecular biological research, particularly in functional genomics are given. The prospects for their use in diagnostics and medicine are discussed.

An effective modification of phosphotriester method for automatic synthesis of DNA and RNA fragments using O-nucleophilic intramolecular catalysis and 2-(azidometil)benzoyl group to protect amino groups of heterocyclic bases of nucleotides is described.

Published data on the main types of reagents capable of introducing covalent interstrand cross links into nucleic acids (NA) are summarized in the present review. The reactivity of cross-linking agents, their preferred binding sites, and methods of determining the cross-link localization in a duplex are discussed. Cell response to DNA cross linking, namely, the blocking of replication and transcription, the initiation of reparation processes, and apoptotic death of the cell, are analyzed, as well as the use of cross-linking reagents in therapy and molecular biology.

Effects of low activity of test-subjects during antiorthostatic hypokinesia on the intragroup structure as well as the semantic composition and the directionality of estimation criteria selected by test-subjects to describe the system of attitudes to oneself and others, and their impact on individual status in the social structure of the group have been studied. It was shown that maintaining of a definite level of individual activity of members is important for the group structure and successful adaptation of test-subjects to the unfavourable factors of hypokinesia. The degree of the ego image integrity and dominance of criteria that infer empathy and positive feelings within the system of individual appraisements determine the social status of a group member. The psychologic signs of a "apet" (psychologically close) and a "outsider" (psychologically strange) members of the group have been revealed.

Approaches to preparing acrylamide and polyacrylamide conjugates with oligonucleotides and some peptide nucleic acid-related DNA mimics are considered. Their physicochemical properties and application to the nucleic acid analysis are discussed.

The synthesis of a series of DNA mimics--peptide nucleic acids, phosphonate analogues of peptide nucleic acids, and their hybrids--is described. The preparative synthesis of the corresponding monomers and the solid phase automated synthesis of oligomers-mimics are developed. Modified phosphonate analogues of peptide nucleic acids, in particular chiral derivatives and those with additional hydroxyl groups in the side chains of the backbone as well as pyrene derivatives of peptide nucleic acids and their phosphonate analogues, are prepared. The ability of the resulting oligomers specifically to hybridize to DNA and RNA complementary chains is studied. It is shown that phosphonate analogues of peptide nucleic acids and their hybrids with peptide nucleic acids can form complexes with the DNA and RNA complementary strands, the stability of the complexes increasing in parallel with the increase in the number of peptide nucleic acid residues in the chain of the mimic. This property, along with good water solubility, provides the precondition for further evaluation of these compounds as antisense and antigene agents.

The Tth DNA polymerase genes from the thermophilic bacterium Thermus thermophilus (strans HB-8 and Tt-111) were cloned and expressed in Escherichia coli cells. The target protein contained a polyhistidine tag at the N-terminus that allowed its single-stage isolation by affinity chromatography on Ni-NTA-agarose. The isolated enzyme displayed both high DNA polymerase and reverse transcriptase activities.

In experiments HUBES and ECOPSY-95 small groups of test-subjects under the conditions of long-term isolation and confinement were noted to split into two fairly stable parts each consisting of two crew members and an "outsider". Strain in the interaction between the "outsider" and the crew tended to grow in acuity as was evidenced by rise in the criteria describing behavior of a small group, and relationship. In both experiments the background for the conflict-threatening strain appeared to be a disintegrated ego-image in both of the "outsiders". Generally, infantilism of ego-image was in hand with hand with low self-assessment. Development by the subjects of ineffective patterns of the attitude system which we consider to be "escape back to the past" and "disintegration" of ego-image overlapped with the most psychologically challenging periods in the experiments, i.e. the start and the end of isolation and confinement, and off-nominal situations.

Dynamics of mental performance, critical periods of adaptation, and cognitive processes were studies in conjunction with extended experimental isolation. Training to simple motor skills was found to be less impeded by prolonged isolation as opposed to operations requiring active involvement of higher psychic functions (memory, concentration, thinking). Experiments HUBES and ECOPSY strengthened the hypothesis concerning possible advent of critical periods in prolonged isolation impacting mental performance. They are the initial period (week 1 in isolation), emergency, arrival of visiting crews, and finishing two weeks. Effect of isolation was not so much evident from changes in absolute meanings of work parameters as from the reduction of their stability in time.

Analyzed were indices of communicative activity of crew members during audio- and computer sessions in order to assess status and effectiveness of communicative interaction of crew with external parties. The investigation gave another evidence that an isolated small group develops "closing" and "internal censorship" in contacts with outer personnel. The conclusion was based on limited in intensity, number, topical spectrum and outer companion preference communication. These appeared to be parallel to egocentrism manifested by claims on supreme concern, sensitivity to speech tones and matter of massages of outer supporters, and attempted manipulation with the outer personnel. In the course of the experiment, the subjects were getting more into the habit to use the information channels for expansion of their emotional contacts and "dumping" emotions. Differential attitude to different duty teams and selective manner of communication underlie differences in the intensity of crew-ground personnel communication.

To verify experimentally the molecular recognition theory, plasmids were constructed that provided the efficient synthesis of hybrid proteins composed of human calcitonin or miniproinsulin, the corresponding antisense peptides, and a histidine-rich metal-binding peptide. A method for isolation of the hybrid proteins by metal-chelating chromatography, cleavage, and renaturation was developed.

Chemicoenzymatic synthesis and cloning of a gene encoding the Fc domain of human immunoglobulin G1 were carried out. The artificial gene was expressed in Escherichia coli cells in plasmid vectors under control of a late T7 promoter. The recombinant protein isolated from the bacterial cells is capable of forming dimers and binding protein A from Staphylococcus aureus.

Plasmid vectors providing a high level expression in Escherichia coli cells of genes for heterologous polypeptides and proteins fused to peptides containing homohistidine clusters have been constructed. The obtained vectors have been used to achieve expression of genes for streptavidin, proinsulin and calcitonin. The hybrid proteins were isolated by affinity chromatography on Ni-agarose columns.

Described is a computerized technique for assessment of psychic performance. It includes a battery of tests in the form of computer games to score recent memory, attention, cognitive power, spatial awareness, and oculomotor orientation. The game-type tests and the possibility of feedback enforce motivation for diagnostic testing. The technique can be advantage for artificial or extreme environments as it can be fulfilled by subjects themselves without assistance of expert.

Recombinant constructs expressing fusion (His)6 movement proteins of two strains (Cruciferous and Vulgare) of tobacco mosaic virus were generated by cloning the PCR-amplified fragments into the pQE-9 vector. The movement proteins containing N-terminal (His)6 affinity tags were purified on a metal chelate adsorbent.

Because of the potential significance of the 'molecular recognition' theory for studies in molecular biology and biotechnology, the theory is worth being examined using methods of chemical-enzymatic gene synthesis, recombinant DNA construction and microbiological peptide synthesis. We therefore undertook the synthesis of human Va18-calcitonin, miniproinsulin, and the corresponding antisense peptides as model compounds. In designing an experimental system the idea was to combine sense and antisense polypeptides into a single chain and to examine their intramolecular interaction. In this paper the chemical-enzymatic synthesis, cloning and expression of the genes for calcitonin, miniproinsulin, the corresponding antisense peptides and their combinations are described. The recombinant DNAs obtained were able to direct in vivo expression of the target polypeptides as hybrid proteins with the IgG-binding domain of the staphylococcal A protein in bacterial cells.

Previously characterized by us barley chloroplast psbA gene, which encodes one of the main Photosystem II components--D1 protein, has been inserted in a set of special plasmid vectors and its expression in vitro and in vivo has been investigated. Experiments on the in vitro expression in the rabbit reticulocyte lysate system revealed a major product with a molecular weight ca. 33.5 kD, which corresponds to the unprocessed D1 barley protein. A lower molecular weight protein (about 29 kD) was also observed. These results are in agreement with the existence of two potential translation start sites in the psbA gene in the same reading frame, the second one starting from Met37 residue. The results fully correlate with the earlier data on the in vitro expression of psbA genes of maize, pea, and tobacco. Experiments on the in vivo expression of psbA gene in E. coli cells with the above constructions also revealed proteins with m. w. about 33.5 and 29 kD. The yield of the target recombinant protein in some cases was about 25-30% of the total E. coli cellular protein. The correspondence of the bands to the desired products was proved by the immunoenzyme analysis with the use of polyclonal antibodies. The data obtained show for the first time the construction of E. coli strains producing recombinant D1 protein of cereals in a high level.

A rapid method for the removal of protecting groups from synthetic oligodeoxyribonucleotides, obtained by the phosphoroamidite and H-phosphonate methods, including the use of hydrazine solutions has been developed. The combination of this procedure with the application of isopropoxyacetyl N-protecting group and oxalyl ester linkage for the first nucleoside attachment to the polymer support allows to reduce to several minutes the time needed for the full oligonucleotide deprotection.

The chemical-enzymatic synthesis of a gene coding for A2B2 repeats of the albumin-binding domain of streptococcal protein G has been accomplished. The codon usage of the natural gene has been modified to adapt an artificial sequence for the efficient translation in E. coli. The gene (238 b.p.) was cloned in the polylinker plasmid pUCL1 and then fused in frame to the 3'-terminus of the gene for the IgG-binding domain of staphylococcal protein A, which was earlier cloned in the expression plasmid pUCL2. A fused polypeptide composed of the E and B domains of protein A and A2B2 repeats of protein G was produced in E. coli cells under the lac promoter control. The resulted product was isolated by affinity chromatography on IgG-sepharose and (or) albumin-sepharose.

Mutagenesis of two previously cloned plant genes, maize storage protein cZ22B1 gene and barley Photosystem II protein D1 gene (psbA), was carried out. To improve the nutritional quality of zein, the DNA region corresponding to the protein sixth alpha-helix rod was substituted by a synthetic segment bearing three codon changes for Lys. Additional stabilization of this helix was achieved by three more codon changes for Glu. By means of oligonucleotide directed mutagenesis five different copies of psbA gene were obtained, bearing single codon change of Ser264 (wild type) for Gly, Ala, Cys, Asn, and Thr, respectively. These constructs can be used for studying functional topography of protein D1 and core region.

The authors review an expert system (ES) of diagnosing the prosthetics quality, elucidate problems of the ES functioning within the framework of the general ES. Special attention is paid to the description of the knowledge and datum surface, to the algorithms of direct and reverse illegible logical data output in taking decisions as to defects of the patient's prosthesis in accordance with the signs observable. The work of the ES is demonstrated with special reference to concrete cases. Analysis is made of the estimates of the sensitivity to the initial data. The ES presents knowledge in depth, being designed in two versions: for practical (individual and mass) prosthetics and training purposes.

In order to determine structures of the barley photosystem II subunits, the following genes have been cloned: psbB, encoding 47 kDa chlorophyll-binding subunit; psbH, encoding 7.7 kDa phosphoprotein; psbE and psbF, encoding 9.3 and 4.4 kDa subunits of the cytochrome b559 apoprotein, respectively; and a fragment of psbC gene, encoding the 43 kDa chlorophyll-binding subunit. The nucleotide sequences of these genes and the deduced amino acid sequences of their products are highly homologous to the corresponding sequences for other plant species.

The authors describe the structure, characteristic features and experience gained with the employment of an expert system for the choice of hip prostheses (in cases of unilateral amputation) and assessment of the prosthetics quality. Word the requirements for hardware and software, present functional subsystems corresponding to certain interrelated stages of prosthetics. A variant has been devised of an appropriate training expert system with a profound representation of intelligence in an object area.

The mechanism of the internucleotide condensation and side-reactions in H-phosphonate approach has been investigated with the help of NMR-spectroscopy. On the basis of the results obtained a modification minimizing side-reactions in the course of the nucleotide component preactivation has been developed. It includes the use of acetonitrile--quinoline (4:1) mixture as the solvent in coupling reactions. The efficiency of the method is illustrated by the synthesis of oligodeoxynucleotides.

To investigate structure-function relationships in zein proteins and to develop an approach to the construction of mutant zeins of improved nutritional qualities, the chemical-enzymatic synthesis of a gene for zein cZ22B1 (22 kd) has been undertaken. This 806-base pair long DNA fragment consists of about 40 synthetic oligonucleotides, mostly 30-60-mers. The synthesis was planned with the use of a universal methodology for the artificial gene construction. The choice of appropriate sites for altering amino acid sequence and the possibility of obtaining by directed mutagenesis of the gene corresponding to modified zeins containing residues Trp and Lys in specified positions of the polypeptide chain is discussed.

Expression of the synthetic gene for human proinsulin in E. coli has been investigated. The proinsulin gene has been expressed directly under the control of a synthetic promoter of phage fd DNA and a promoter of tryptophan operon, or using fusions with fragments of some bacterial proteins. These fusions gave insoluble polypeptide products amounting to 20-30% of total cellular protein. The scheme for isolating proinsulin from bacterial cells was developed. Proinsulin was cleaved from leader polypeptides by treatment with cyanogen bromide and converted into human insulin.

A procedure has been designed for changing specific nucleotides in a DNA sequence with efficiency. The method involves the use of the specially constructed cloning vectors pBRS1, pHS1, and pHS2. These plasmids are derivatives of pBR322 in which the EcoRI-HindIII region has been replaced by synthetic duplexes carrying SmaI, HpaI and XhoI sites, in addition to EcoRI and HindIII sites. The DNA fragment to be mutagenized is cloned in pHS1 (or pBRS1, or pHS2) using restriction sites close to the SmaI and HpaI sites. The recombinant plasmid obtained is digested with one of these enzymes to produce double-stranded DNA with blunt ends. This linear DNA is a substrate for exonuclease III (or T4 DNA polymerase). The digestion under controlled conditions produces duplex with protruding single-stranded 5'-regions which include the site of the desired mutation. The synthesis of DNA by DNA-polymerase I (Klenow's fragment), primed in part by the synthetic oligonucleotide containing the desired mutation, leads to the linear heteroduplex. The closed circular heteroduplex is formed by ligation. After transformation into E. coli, DNA replication generates homoduplexes, some of which contain the mutation. Colony hybridization with the same 32P-labeled oligonucleotide is used to select mutants. The yield of the mutants is 10-20%. This technique can be extended to replicative form of M13 vectors. It can be also applied to any DNA sequence which has a unique site of restriction endonuclease generating blunt ends.

The chemical-enzymatic synthesis of a gene for IgG-binding fragment of the staphylococcal protein A has been carried out. The design of the gene, which consists of signal peptide and modified E and B domains, and strategy of the synthesis provided possibility of various degrees of polymerization of the gene fragment coding for B domain and of the whole gene. Several protein A-like polypeptides composed of the leader sequence, E domain and 1 to 4 copies of B domain were produced in E. coli cells under the lac promoter control.

Using oligonucleotide-directed mutagenesis and chemical-enzymatic DNA synthesis, genes for A and B insulin chains, C-peptide and Tyr-C-peptide have been constructed starting from synthetic gene for human proinsulin synthesized earlier. The genes for human preproinsulin, mini-proinsulin, single-chain insulin and their modifications were also synthesized. The constructions obtained were cloned in plasmid vectors.

A useful and efficient approach to the synthesis of DNA duplexes of practically unlimited length has been developed. The proposed methodology is based on the use of temporary restriction sites for subcloning and assembling the segments of the desired DNA. It allows the utilization of chemically synthesized oligonucleotides of various length (from 10- to 100-mers) for the duplex construction. The application of this approach to the synthesis of a gene for the functionally active bacteriorhodopsin fragment is described.

The psbD gene for the membrane polypeptide D2 has been isolated from barley chloroplast DNA and its sequence, along with the flanking regions, has been determined. The 3'-end of the psbD gene is overlapped with a 50 bp stretch of a second open reading frame which belongs to the psbC gene encoding the P6 protein of photosystem II.

With the use of synthetic DNA fragments, a set of new plasmid vectors has been obtained. The vectors provided high level expression of peptides and small proteins in E. coli as fusions with fragments of beta-galactosidase of various length. These vectors were used to achieve expression of a synthetic gene for a functionally active fragment of bacteriorhodopsin. The yields of hybrid proteins consisting of beta-galactosidase and bacteriorhodopsin fragments were in the range of 5-30% from the total amount of cellular protein.

The sequence of a stretch of the barley chloroplast DNA containing psbA gene with flanking regions and the deduced amino acid sequence of the photosystem II QB protein have been determined. Comparison of the psbA and QB sequences from barley and some dicotyledonous species shows them to be highly conserved. The sequences of the regions which flank the probable transcription initiation site for psbA gene of barley are identical to the corresponding sequences of wheat.