[Peptide nucleic acids and their phosphonate analogues: synthesis and hybridization characteristics]. / Peptido-nukleinovye kisloty i ikh fosfonatnye analogi: sintez i gibridizatsionnye svoistva.

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.

[Biosynthesis of human calcitonin and mini-proinsulin in bacterial cells in the form of hybrid proteins with corresponding antisense peptides and a metal-binding peptide]. / Biosintez v bakterial'nykh kletkakh kal'tsitonina i mini-proinsulina cheloveka v vide gibridnykh belkov s sootvetstvuiushchimi antismyslovymi peptidami i metallsviazyvaiushchim peptidom.

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.

[Synthesis and cloning of genes for the antisense peptides for human calcitonin and miniproinsulin]. / Sintez i klonirovanie genov antismyslovykh peptidov kal'tsitonina i miniproinsulina cheloveka.

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.

[Construction of a family of artificial genes of human insulin]. / Konstruirovanie semeistva iskusstvennykh genov insulina cheloveka.

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.

[Synthesis of human proinsulin in Escherichia coli cells]. / Sintez proinsulina cheloveka v kletkakh Escherichia coli.

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.

[Synthesis and expression of an artificial gene of an IgG-binding fragment of protein A from Staphylococcus aureus]. / Sintez i ékspressiia iskusstvennogo gena IgG-sviazyvaiushchego fragmenta bela A Staphylococcus aureus.

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.

[Construction of promoter-probe vectors on the basis of a modified beta-galactosidase gene of Escherichia coli]. / Konstruirovanie promotornprobnykh vektorov na osnove modifitsirovannogo gena beta-galaktozidazy.

Plasmid-based promoter-probe vectors pPV4 and pPV5 have been constructed which are useful for comparing the relative efficiencies of bacterial promoters. The vectors utilize the beta-galactosidase (lacZ) gene of E. coli as an indicator gene. The latter was modified using synthetic DNA fragments. The promotor-probe system contains the ampicillin resistance gene and the origin of replication of plasmid pBR322. The plasmids pPV4 and pPV5 carry clustered unique restriction sites usable for promoter insertions, and SD sequence. A synthetic DNA fragment corresponding to transcription terminator was inserted downstream the lacZ gene. Presence of the terminator made it possible to clone strong promoters controlling transcription of the lacZ gene. To prevent any undesired promotor effect, the plasmid pPV5 has also second synthetic terminator upstream from the polylinker sequence. Using this promoter-probe system, relative efficiencies of a series of synthetic promoters, including PL promoter of phage lambda and its mutant, gene X promotor of phage fd and several model statistic promoters, have been compared.

[Synthesis and cloning of artificial zein genes]. / Sintez i klonirovanie iskusstvennykh genov zeinov.

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.

[General approach to the engineering of synthetic DNA]. / Obshchii podkhod k konstruirovaniiu sinteticheskikh DNK.

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.