Structural variations of germanosilicate glasses with change in modifier cation type or Ge/Si ratio.

Raman spectroscopy was used to study glasses of three-component systems M2O-SiO2-GeO2, where M = Li, Na, K. Following the decomposition of spectra into component lines, the main structural units of both the germanate and silicate networks were determined according to peak-fitting result. With addition of germanium oxide, a change in the structure of the silicate network system was observed. In revealing highly-coordinated germanium atoms in all synthesised glasses, the study also established the dependence of their quantity on GeO2 content and modifier cation type.

Aggregation of Influenza A Virus Nuclear Export Protein.

Influenza A virus nuclear export protein (NEP) plays an important role in the viral life cycle. Recombinant NEP proteins containing (His)6-tag at either N- or C-terminus were obtained by heterologous expression in Escherichia coli cells and their high propensity for aggregation was demonstrated. Dynamic light scattering technique was used to study the kinetics and properties of NEP aggregation in solutions under different conditions (pH, ionic strength, presence of low-molecular-weight additives and organic solvents). Using atomic force microscopy, the predominance of spherical aggregates in all examined NEP preparations was shown, with some amyloid-like structures being observed in the case of NEP-C protein. A number of structure prediction programs were used to identify aggregation-prone regions in the NEP structure. All-atom molecular dynamics simulations indicate a high rate of NEP molecule aggregation and reveal the regions preferentially involved in the intermolecular contacts that are located at the edges of the rod-like protein molecule. Our results suggest that NEP aggregation is determined by different types of interactions and represents an intrinsic property of the protein that appears to be necessary for its functioning in vivo.

Heterologous Expression and Isolation of Influenza A Virus Nuclear Export Protein NEP.

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N- or C-terminal (His)6-tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

A hypothetical hierarchical mechanism of the self-assembly of the Escherichia coli RNA polymerase σ(70) subunit.

Diverse morphology of aggregates of amyloidogenic proteins has been attracting much attention in the last few years, and there is still no complete understanding of the relationships between various types of aggregates. In this work, we propose the model, which universally explains the formation of morphologically different (wormlike and rodlike) aggregates on the example of a σ(70) subunit of RNA polymerase, which has been recently shown to form amyloid fibrils. Aggregates were studied using AFM in solution and depolarized dynamic light scattering. The obtained results demonstrate comparably low Young's moduli of the wormlike structures (7.8-12.3 MPa) indicating less structured aggregation of monomeric proteins than that typical for ß-sheet formation. To shed light on the molecular interaction of the protein during the aggregation, early stages of fibrillization of the σ(70) subunit were modeled using all-atom molecular dynamics. Simulations have shown that the σ(70) subunit is able to form quasi-symmetric extended dimers, which may further interact with each other and grow linearly. The proposed general model explains different pathways of σ(70) subunit aggregation and may be valid for other amyloid proteins.

[Investigation of the Dependence of Escherichia coli RNA Polymerase σ70-Subunit Structure on Ionic Strength by Molecular Dynamics Simulation Method].

The σ70-subunit of E. coli RNA polymerase (a small protein, being a part of RNA holoenzyme, and responsible for initiation of transcription of constitutive genes) is modeled at different ionic strengths. Two variants of the location of C-end domain 4 are obtained. At low ionic strength domain 4 interacts with the region of high negative charge 190-210 AK within NCR domain. At high ionic strength this region was screened and domain 4 was free and set away from domain NCR. We suppose that this leads to the increase in polymerization rate. Simulation data do not confirm any hypothesis about a self-inhibition mechanism.

Investigation of activity of recombinant mengovirus RNA-dependent RNA polymerase and its mutants.

The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryptophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3'-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme.

Purification of core enzyme of Escherichia coli RNA polymerase by affinity chromatography.

A method for isolation of a highly purified preparation of E. coli RNA polymerase core enzyme was developed based on IMPACT technology and dissociation of the RNA polymerase complex with sigma(70) subunit. Washing of the immobilized RNA polymerase with 5-10 mM solution of glutamate (pH 5.0-5.5) completely removed the sigma(70) subunit from the holoenzyme and decreased amounts of protein admixtures. The possibility of reconstruction of the RNA polymerase holoenzyme directly on the affinity column was demonstrated. Activities of the resulting RNAP core enzyme preparations were tested by in vitro transcription. Some amino acids and their mixtures were shown to influence the in vitro transcription. The findings indicate that changes in the transcription efficiency in the presence of amino acids should be associated with a specific destruction of the interaction between sigma(70) subunit and the core enzyme.

A system for heterologous expression and isolation of Escherichia coli RNA polymerase and its components.

A set of plasmid vectors for expression of all major Escherichia coli RNA polymerase subunits as fusion proteins with intein- and chitin-binding domains, allowing protein purification in accordance with IMPACT technology, was constructed. It is demonstrated that the fusion subunits alpha, beta or beta' in conjunction with the natural subunits alpha, beta, beta', and sigma can participate in RNA polymerase assembly in vivo, providing affinity-based isolation of the enzyme. Functional activity of the enzyme preparations was demonstrated in the experiments on in vitro transcription and promoter complex formation. With the use of IMPACT technology, sigma(70) subunit can be isolated as an individual protein without admixture of RNA polymerase.

[Features of replicating synthetic oligonucleotides with non-native chains]. / Osobennosti replikatsii sinteticheskikh oligonukleotidov s neprirodnymizven'iami.

The in vitro replication of synthetic oligodeoxyribonucleotides carrying internucleotide polyphosphate groups or alkanediol "spacers" of various sizes with the use of various DNA polymerases has been studied. All modifications, except for the diphosphate group, almost completely block the polymerization process. In the case of AMV reverse transcriptase, Taq and T7 DNA polymerases and also the Klenow fragment of E. coli DNA polymerase I, a template-independent addition of a nucleotide at the 3' end of the incomplete replica was observed. T4 DNA polymerase, displaying the strongest 3'-5' exonuclease activity among the polymerases studied, did not incorporate additional nucleotides. The use of oligonucleotides with non-nucleotide inserts as primers in polymerase chain reaction (PCR) allows to obtain DNA copies with protruding 5'-termini, suitable for hybridisation analysis.

[A model prokaryotic promotor. III. Cloning and study of functional activity in vivo]. / Model'nyi prokarioticheskii promotor. III. Klonirovanie i issledovanie funktsional'noi aktivnosti in vivo.

The efficiency of two schemes of oligonucleotide-directed insertion mutagenesis was studied in comparison with standard cloning of DNA duplexes based on blunt-end ligation. Using new approaches, the 30-bp consensus-like prokaryotic promoter was inserted in proper orientation in a promoter-testing plasmid at an almost 100% frequency. Data on the marker gal operon expression and S1-nuclease mapping of the transcription initiation points indicate formation of an active promoter in the region of the insertion.

[Design of mixed polymers based on DNA fragments with consensus promotor elements, separated by nonnucleotide segments]. / Konstruirovanie smeshannykh polimerov na osnove fragmentov DNK s konsensusnymi élementami promotorov, razdelennykh nenukleotidnymi uchastkami.

Mixed oligomers, representing oligonucleotides connected with long non-nucleotide spacers, have been synthesized using phosphoramidite chemistry. The oligonucleotide moieties of the mixed oligomers fully or partially correspond in the structure to the consensus elements of -35 (TTGACA) and -10 (TATAATG) regions of prokaryotic promoters. The non-nucleotide spacers, approximating in size 17-membered DNA fragments, were synthesized using phosphoramidite derivatives of polyethylene glycol (PEG600), tetraethylene glycol or dodecanediol. It is shown that the oligonucleotide moieties of the mixed oligomers can form "normal" DNA like antiparallel complementary complexes, being the substrates of T4 DNA ligase. To obtain the DNA-like polymers with alternating natural and non-natural regions or cyclic structures, the enzymatic ligation of different complexes of the oligomers synthesized was studied.

[A model prokaryotic promotor. II. Function of designs with mutations in the "-35" region]. / Model'nyi prokarioticheskii promotor. II. Funktsionirovanie konstruktsii s mutatsiiami v oblasti "-35".

A series of DNA duplexes corresponding to the E. coli consensus promoter with random hexanucleotides in the -35 region have been synthesized and characterized. The library of recombinant plasmids with synthetic promoter-like inserts, oriented in the direction of the marker gal operon of the initial vector, have been obtained. Analysis of the library on indicator medium and S1 mapping of the in vivo transcription initiation points in several plasmids have shown that most constructions exhibit promoter properties, and the structures of their -35 regions may be varied.

[Chemical reactions in double-helical nucleic acids. XVI. Synthesis of DNA-duplexes containing regularly repeating transverse covalent cross- links]. / Khimicheskie reaktsii v dvuspiral'nykh nukleinovykh kislotakh. XVI. Sintez DNK-dupleksov, soderzhashchikh reguliarno povtoriaiushchiesia poperechnye kovalentnye sshivki.

Two self-complementary decadeoxyribonucleotides TAATGC*ATTA (where C* is a derivative of 5-methyl cytosine with a carboxy- or aminofunction attached through a spacer to the exocyclic amino group) were synthesized. Carbodiimide induced condensation of the amino and carboxyl groups in the opposite strands to give the crosslinks with a yield up to 20%. Cross-linking of two opposite strands in the duplex formed by the self-complementary aliphatic amino group-containing decanucleotide was performed with the use of glutaric aldehyde with a similar efficiency. The structure of the dimers obtained and position of the crosslinks were confirmed by the Maxam--Gilbert method. Efficiencies of the T4 DNA ligase-induced polycondensations of the double-stranded modified decanucleotides and of the cross-linked products differed significantly.

[Features of connecting short oligonucleotides with phage T4 DNA ligase]. / Osobennosti soedineniia korotkikh oligonukleotidov DNK-ligazoi faga T4.

To understand the influence of different factors on the efficiency of T4 DNA ligase-catalyzed connection of oligonucleotides, a comparative study of polycondensation of AT-containing concatemer octanucleotides (some of them potentially can form "parallel" duplexes) has been undertaken. It has been shown that the size of substrate duplex is the most significant factor, while the sequence of oligonucleotides influences the enzymatic reaction indirectly, by determining the thermal stability of double-stranded complexes. In practice, none of the octanucleotides under study does form "parallel" duplexes.

[An effective method for site-directed mutagenesis in plasmids and cloning single-stranded DNA fragments]. / Effektivnyi metod napravlennogo vvedeniia mutatsii v plazmidy i klonirovaniia odnotiazhevykh fragmentov DNA.

A three primer variant of the earlier devised oligonucleotide-directed mutagenesis in plasmids is described, useful also for the fast cloning of single-stranded DNA products of the asymmetric polymerase chain reaction (PCR). Using this method for plasmid pHD-001-14-11, a 59 b. p. deletion and a 7 b. p. insertion were simultaneously introduced at 81% frequency, and the PCR-copied phage fd transcription terminator (26 b. p.) was inserted with the yield of 67%.