[Application of BODIPY-trimethylmelamine conjugate for DNA cross-linking in vitro].

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.

[Methoxymethyl and (p-nitrobenzyloxy)methyl groups in the synthesis of oligoribonucleotides by the phosphotriester method].

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.

[Monomers containing 2'-o-alkoxymethyl groups as synthons for the synthesis of oligoribonucleotides by the phosphotriester method].

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.

Cross-linked nucleic acids: isolation, structure, and biological role.

This review includes literature data on main types of reagents inducing covalent cross-links of nucleic acids. Reactivity of cross-linking agents, preferable sites for their binding, and methods for determination of location of cross-links in duplex are discussed. Biological responses of cells to cross-linking in nucleic acids, i.e. replication and transcription blocking, onset of repair processes, and apoptotic cell death are considered, as well as application of cross-linking reagents as medicinal drugs for solving molecular-biological problems.

[DNA mimics on the base of pyrrolidine and hydroxyproline].

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.

[Cross-linked nucleic acids: formation, structure, and biological function].

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.

[N-azidomethylbenzoyl blocking group in the phosphotriester synthesis of oligonucleotides].

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.

[An azidomethyl protective group in the synthesis of oligoribonucleotides by the phosphotriester method].

A rapid and effective method of an automatic oligoribonucleotide synthesis alternative to the phosphoroamidite one was developed based on the phosphotriester approach of internucleotide bond formation under intramolecular O-nucleophilic catalysis and the use of an azidomethyl group for protection of a nucleotide 2'-hydroxyl function.

Synthesis of polyacrylamides N-substituted with PNA-like oligonucleotide mimics for molecular diagnostic applications.

Two types of oligonucleotide mimics relative to peptide nucleic acids (PNAs) were tested as probes in nucleic acid hybridisation assays based on polyacrylamide technology. One type of mimic oligomers represented a chimera constructed of PNA and phosphono-PNA (pPNA) monomers, and the other one contained pPNA residues alternating with PNA-like monomers on the base of trans -4-hydroxy-L-proline (HypNA). A chemistry providing efficient and specific covalent attachment of these DNA mimics to acrylamide polymers using a convenient approach based on the co-polymerisation of acrylamide and some reactive acrylic acid derivatives with oligomers bearing 5'- or 3'-terminal acrylamide groups has been developed. A comparative study of polyacrylamide conjugates with oligonucleotides and mimic oligomers demonstrated the suitability and high potential of PNA-pPNA and HypNA-pPNA chimeras as sequence-specific probes in capture and detection of target nucleic acid fragments to serve current forms of DNA arrays.

Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach.

An effective procedure for specific determination of the cap structure at the 5'-terminus of mRNA and for isolation of the corresponding full-length cDNA has been developed. The procedure involves covalent attachment of an oligonucleotide template extender to the 5'-cap structure of mRNA followed by RT-PCR using M-MLV SuperScript II reverse transcriptase. In the course of reverse transcription, the enzyme 'jumps over' the cap structure and includes the sequence complementary to the oligonucleotide template extender into the 3'-end of the first cDNA strand. The cap-jumping method was successfully tested using some mammalian cellular mRNAs, genomic RNAs of tobacco mosaic virus (TMV) U1 and the recently isolated crucifer-infecting tobamovirus. Moreover, cDNA products corresponding to the genomic tobamovirus RNA were obtained from total RNA extracted from tobacco plants infected by crucifer-infecting tobamovirus or tobacco mosaic virus. Using the cap-jumping method, we have shown for the first time that genomic crucifer-infecting tobamovirus (crTMV) RNA contains a 5'-cap structure. This improved method can be recommended for the construction of full-length and 5'-end enriched cDNA libraries, identification of capped RNAs and determination of their 5'-terminal sequences.

Synthesis of DNA coding for human proinsulin.

A chemical-enzymatic synthesis of 271- and 286-bp DNA duplexes, each of which contains the entire sequence coding for human proinsulin has been accomplished. In addition to the coding sequence, the 271-bp fragment carries translation initiation and termination signals plus EcoRI-HindIII restriction enzyme sites for insertion into an appropriate plasmid vector. The 286-bp fragment also contains a Shine-Dalgarno (SD) sequence preceding an ATG codon. Employing the 286-bp polynucleotide, the 568-bp tandem proinsulin gene has been obtained. The synthesis of these DNA fragments involved preparation of 42 oligonucleotides by a rapid N-methylimidazolide phosphotriester method and enzymatic conversion of the oligonucleotides into the gene subfragments, which were cloned separately and fused to yield the desired DNAs coding for proinsulin. The proinsulin gene fragments were cloned in Escherichia coli and shown to have the correct sequences.

Convenient modification of the method for oligonucleotide-directed in vitro mutagenesis of cloned DNA.

A new modification of the oligonucleotide-mediated mutagenesis technique has been developed. The proposed methodology has been used to produce specific base changes in the double-stranded plasmid DNA. For this purpose, special cloning vectors have been constructed using the synthetic oligodeoxyribonucleotides. The developed method allows the production of mutant DNA from those of the wild-type with a yield of 10-20%.

Complete nucleotide sequence and genome organization of a tobamovirus infecting cruciferae plants.

Genomic RNA sequence of a tobamovirus infecting cruciferae plants (cr-TMV) was determined. The RNA is composed of 6312 nucleotides and contains four ORFs encoding the proteins of 122K (ORF1), 178K (ORF2), 29K (ORF3) and 18K (capsid protein, ORF4). ORF4 overlaps ORF3 by 74 nucleotides and the overlapping region can be folded into a stable hairpin structure. The 3'-terminal region of the cr-TMV RNA preceding the tRNA-like structure was shown to form six potentially stable pseudoknots.

Expression of the barley psbA gene in Escherichia coli yields a functional in vitro photosystem II protein D1.

The barley chloroplast psbA gene encoding D1 protein, one of the main photosystem II components, has been over-expressed in E. coli cells. The existance of two in vivo expression products, a protein with M(r) about 33.5 kDa, corresponding to the full-length precursor of the 32 kDa D1 mature form, and a truncated 29 kDa polypeptide was revealed. A modified D1 protein containing six histidine residues at the carboxy-terminus was also obtained. After isolation and renaturation, the ability of the recombinant D1 protein to bind atrazine and pigments from barley thylakoids was demonstrated.

Rapid synthesis of oligonucleotides by the phosphotriester method.

An efficient phosphotriester methodology based on the use of condensing agents in the presence of several O-nucleophilic catalysts has been developed.

Sequence specificity of alternating hydroyprolyl/phosphono peptide nucleic acids against zebrafish embryo mRNAs.

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.

Subject's perceptions of the crew interaction dynamics under prolonged isolation.

BACKGROUND: The interactions of individuals participating in space simulation studies can give us valuable information about ways that space crew members relate with one another. A promising method of investigating such interactions is based on the Kelly repertory grid technique. HYPOTHESES: We predicted that in a space simulation crew: 1) one's personal self-concept would become closer to concepts about other crew members in a cohesive crew, and vice-versa, and 2) the similarity of one's ego images in the past, present and future influences that person's position in the group. METHOD: Crews from two isolation studies lasting 135 and 90 days at the Institute for Biomedical Problems in Moscow were evaluated using a modification of the Kelly repertory grid technique. Members assessed each other on a monthly basis and during off-nominal simulated docking periods. RESULTS: During both isolations, crew members were unsuccessful in making their personal self-concepts move closer to their concepts about fellow crew members. Crew disintegration resulted, with one member becoming an outsider whose personality was characterized by a disintegrated ego-image and an infantile type of the present ego. CONCLUSIONS: In small isolated groups, failure to make personal self-concepts become more like concepts about other crew members can lead to group disintegration and tension and can result in the appearance of an outsider who has a disintegrated ego-image and an infantile present ego.

Content analysis of the crew communication with external communicants under prolonged isolation.

BACKGROUND: The communication between space crews and outside monitoring personnel in Mission Control gives us valuable information about crewmember psychological health. Studying the formal parameters of similar communication during ground-based space simulation missions helps us understand the psychological climate of confined groups over time, and this information can be applied to long-term space missions. HYPOTHESES: We hypothesized that psychological closing and information filtration will be observed in a confined crew as the mission progresses and will differ by the type of communication (e.g., audio vs. computer-generated) with the outside monitors in the simulated Mission Control (MC). In addition, communication patterns will vary with the specific group of people on-duty outside. METHOD: Audio and computer communications between space simulation crews and MC teams were analyzed during two isolation studies lasting 135 and 90 d at the Institute for Biomedical Problems in Moscow. A number of temporal, content and quantitative parameters of verbal activity were examined. RESULTS: The total intensity of communication dropped after month 1 of the missions. The Commander had the highest intensity of communication. There was evidence for the presence of psychological closing and information filtration in the crews over time, with some variation based on type of communication. Communication patterns differed with different outside teams. CONCLUSIONS: The communication between confined groups and outside monitoring personnel is affected by psychological closing and information filtration and by the make-up of the teams that comprise the monitoring groups.

[Conjugates of polyacrylamide with oligonucleotides and their mimetics for diagnostic purposes]. / Kon''iugaty poliakrilamida s oligonukleotidami i ikh mimetikami dlia diagnosticheskikh tselei.

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.

[Modification of H-phosphonate method of oligonucleotide synthesis on polymer support]. / Modifikatsiia H-fosfonatnogo metoda sinteza oligonukleotidov na polimernykh nositeliakh.

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.