[2'fluoro derivatives of nucleosides as substrates of viral replicative nucleotide polymerases].

Substrate specificities of three viral replicative polymerases of different origins (HIV reverse transcriptase, hepatitis C virus RNA polymerase, and herpes virus DNA polymerase) towards 2'F-NTP were studied. Activated DNA, polyA-oligoUs and (2'F-A)20-oligoU6-complexes were used as templates. It was shown that all DNA polymerases studied can incorporate 2'F-NMP into the 3'-end of primer-template complexes. HIV reverse transcriptase and herpes virus DNA polymerase can elongate synthesis with both dNTP and 2'F-NTP. Homopolymer (2'F-A)20 can serve as a template for polymerization of both UTP and 2'F-UTP,-catalyzed by hepatitis C virus polymerase although with efficacy about 5 to 10-fold lower in comparison with natural primertemplate complex. Pyrophosphorolysis reaction of 2'F-CMP residue at 3'-end of primer catalyzed with HIV reverse transcriptase is going by two orders of magnitude less effective if compared with natural dNMP residue at the same system.

[Secretion of recombinant human epidermal growth factor into the periplasm in Escherichia coli cells]. / Sekretsiia v periplazmu rekombinantnogo épidermal'nogo faktora rosta cheloveka v kletkakh Escherichia coli.

Secretion vectors were constructed in which a synthetic gene of human epidermal growth factor (hEGF) joined with a gene coding for the leader peptide to one of the E. coli outer membrane major proteins (OmpF) is controlled by tac promoter. The increase of the hEGF yield was achieved by the multiplication of the gene copies. The hEGF in bacterial cells was secreted into periplasm. The recombinant protein was isolated by means of reverse phase chromatography as almost homogenous preparation (greater than 98%), the yield being 7 mg/l bacterial culture. The sequence of twenty-five N-terminal amino acid residues of the isolated hEGF coincided with that of the natural protein. The preparation proved to be biologically active.

[Expression of a synthetic gene for human interleukin-4 in E. coli cells. Preparation of a biologically active protein]. / Ekspressiia sinteticheskogo gena interleikina-4 cheloveka v kletkakh E. coli. Poluchenie biologicheski aktivnogo belka.

Expression E. coli plasmid were constructed in which the human interleukin-4 (hIL4) synthetic gene is controlled by tac promoter. The expression level of the gene depends on the distance between RBS and the initial codon ATG, with the maximal production in case of the nine base pair distance. The recombinant protein, accumulated in the inclusion bodies, was solubilized, renaturated, and purified to homogeneous, biologically active preparation, the yield being 2 mg/g wet cells.

[Isolation of recombinant interleukin-3 produced by E. coli]. / Vydelenie rekombinantnogo interleikina-3, produtsiruemogo E. coli.

A synthetic gene coding for human interleukin-3 (hIL3) was cloned in the plasmid pTE2IL3, the gene expression being controlled by the phage fd PVIII promotor and the phage T7 gene 10 translational enhancer. Under constitutive biosynthesis conditions in E. coli, the accumulation of recombinant hIL3 (in the inclusion bodies) was up to 30-40% of the total cell protein. An effective procedure of the hIL3 isolation is suggested. The hIL3 was solubilized in 5 M guanidinium chloride, renaturated and purified to homogeneity by a single chromatographic step. The protein's yield was 34 mg/g wet cells. The isolated hIL3 showed a specific biological activity.

[Chemical-enzymatic synthesis of the amplifier of the T7 phage gene 10 and function of its structural elements]. / Khimiko-fermentativnyi sintez usilitelia transliatsii gena 10 faga T7 i funktsiia élementov ego struktury.

The translational enhancer (TREN) sequence of the phage T7 gene 10 (in full and also its proximal or distal parts) have been obtained by chemical-enzymatic synthesis and cloned into the plasmids immediately before the human interleukin 3 (hIL3) artificial gene. Expression levels of the hIL3 gene in E. coli in these constructions show that the region controlling the specific activity is placed in distal part of TREN more than 40 nucleotides upstream from the initiation codon.

[H-phosphonate method in the synthesis of structural gene of human interleukin-4]. / H-fosfonatnyi metod v sinteze strukturnogo gena interleikina-4 cheloveka.

A modified H-phosphonate method was used to synthesize 32 oligodeoxyribonucleotides ranging in length from 23 to 28, which were enzymatically joined together to give the human interleukin 4 gene. The high degree of the oligonucleotide purity, achieved through the application of anion-exchange and reverse phase HPLC, ensures the high percentage of the desired sequence (about 75%) in the cloned DNA.

[N-phosphonate synthesis of oligodeoxyribonucleotides with the use of a p-nitrophenylethyl blocking group]. / N-foxfonatnyi sintez oligodezoksiribonukleotidov s ispol'zovaniem p-nitrofenilétil'noi zashchitnoi gruppy.

p-Nitrophenylethyl blocking group was used to protect the endocyclic imido groups of guanine and thymine nucleoside 3'-H-phosphonates employed in the H-phosphonate synthesis of a large number of oligodeoxyribonucleotides varying in length from 8 to 45 units. A combination of the fully protected monomers with a condensing agent, pivaloyl chloride or mesitylenesulphonyl-3-nitro-1,2,4-triazole, provides a rapid and effective synthesis of long oligonucleotides.

[Analogs of natural nucleosides and their 5'-triphosphates carrying the amino group in 3'-position--highly effective inhibitors of replication, repair and transcription]. / Analogi prirodnykh nukleozidov i ikh 5'-trifosfatov, nesushchie v 3'-polozhenii aminogruppu,--vysokoéffektivnye ingibitory replikatsii, reparatsii i transkriptsii.

The new type of DNA and RNA synthesis inhibitors is found. These compounds are active in synthesis, catalyzed by bacterial DNA and RNA polymerases and by eukaryotic DNA polymerases in vitro and in DNA replication in vivo.

[Model substates and inhibitors of the peptidyltransferase center of ribosomes. Conformational possibilities in aqueous solution]. / Model'nye substraty i ingibitory peptidiltransferaznogo tsentra ribosom. Konformatsionnye vozmozhnosti v vodnum rastvore.

The constants of sin-anti-equilibrium in aqueous solution of model inhibitors of peptidyltransferase center of ribosomes: 3'-amino-3'-deoxadenosine-5'-phosphate, 3'-N-glycinamido-3'-deoxyadenosine, 3'-(N-formyl-L-methionynamido)-3'-deoxyadenosine-5'-phosphate and 3'-(n-formylglycinamido)-3'-deoxyadenosine-5'-phosphate were determined using the measurements of spin-lattice relaxation times. All compounds have similar conformation possibilities of the nucleotide component. The possibilities of correlation between the biological activity of these compounds and their conformation in aqueous solution are discussed.

[Peptidyl transferase center of ribosomes. I. Difference in the substrate specificity of the acceptor and donor portions]. / Peptidiltransferaznyi tsentr ribosom. I. Razlichie v substratnoi spetsifichnosti aktseptornogo i donornogo uchastkov.

Some model substrates of the peptidyl transferase centre of E. coli MRE-600 ribosomes were synthesised and tested in a cell-free system without a template. In these substances the nucleic bases were linked covalently with the ribose residue or had a limited rotation about the glycosidic bond. 3'(2')-O-(N-formylmethionyl)-8-bromoadenosine 5'-phosphate and 3'(2')-O-phenylalanyl-8,5'-anhydro-8-mercaptoadenosine were shown to possess a high peptide donor and acceptor activity correspondingly. Contrary to that 3'(2')-O-phenylalanyl-8-bromoadenosine was practically inactive as a peptide acceptor and 3'(2')-O-(N-formylmethionyl)-8,5'-anhydro-8-mercaptoadenosine had no peptide donor activity at all. PMR and CD spectra of the compounds synthesised were investigated. The significance of conformation of the model substrates on their activity is discussed.