Identification and mapping of ten new potential insulators in the FXYD5-COX7A1 region of human chromosome 19q13.12.

A positive-negative selection system revealed 10 potential insulators able to block enhancer interaction with promoter in the 10(6) bp human chromosome 19 region between genes FXYD5 and COX7A1. Relative positions of insulators and genes are in accord with the hypothesis that insulators subdivide genomic DNA into independently regulated loop domains.

[Study of transactivation effect on transcription by Tat-TAR-system of human immunodeficiency virus type 1 (HIV-1) in non-lymphoid cells HEK293 and Calu-1].

An important problem in the development of gene therapy approaches in oncology is the necessity of using promoters providing specific and high level of gene expression in tumor cells. To solve this problem, we used inducible system of gene expression regulation (Tat-TAR-system), which is utilized by human immunodeficiency virus (HIV). tat and tk-HSV genes, as well as a fragment of LTR HIV-1, were cloned in the retrovirus vector, tk-HSV gene was under control of the LTR HIV-1 fragment. Potential capacity of these constructions for transactivating tk-HSV gene transcription was studied. Basal expression level of this gene was defined in transient transfection of HEK293 cells. It was shown that specific transactivation of the tk-HSV gene was controlled by the LTR HIV-1 fragment in lung carcinoma cells Calu-1, permanently transfected by the tat gene construction. The effect of transactivation of tk-HSV transcription in Tat-TAR-system was demonstrated in Calu-1 cells in conditions of control of cancer-specific tat gene over BIRC5 promoter.

[Conservation of the regulatory elements implicated in the control of the rpsB-tsf operon expression in gamma-proteobacteria].

In eubacteria, the rpsB-tsf operon encodes two essential components of translational apparatus, ribosomal protein (r-protein) S2 and elongation factor Ts. Recently, we located the promoter region of the Escherichia coli rpsB-tsf operon and demonstrated that both rpsB and tsf genes are negatively regulated by r-protein S2 at the translational level. In this paper, we present data of phylogenetic analysis showing high conservation of both the promoter signature and the structure of the 5'-untranslated region (5'-UTR) of the rpsB mRNA in gamma-proteobacteria. Despite the difference in length and overall primary structure of the rpsB 5'-UTRs for various representatives of this bacterial phylum, several short regions within the 5'-UTRs appeared to be universally conserved, implying their participation in the expression regulation. Phylogenetic predictions have been experimentally confirmed. We show here that the presumable rpsB promoter regions from Yersinia pestis, Haemophilus influenzae and Pseudomonas aeruginosa are able to drive transcription of the lacZ -reporter in E. coli and that the corresponding rpsB 5'-UTRs are subjected to autogenous repression by r-protein S2 in vivo.

[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.

[Chemical synthesis and cloning of the human epidermal growth factor gene]. / Khimicheskii sintez i klonirovanie gena épidermal'nogo faktora rosta cheloveka.

The solid-phase phosphotriester method was used to synthesise 24 oligodeoxyribonucleotides, which were enzymatically joined together to give the human epidermal growth factor gene and its analogue containing Leu codon in position 21. The primary structure of the cloned genes were confirmed by the Maxam-Gilbert technique. It is demonstrated that the high purity degree of oligonucleotides allows to synthesise and clone genes without purification of intermediate fragments.

[Inhibition of influenza virus A RNA-polymerase activity by various 3'-amino-3'-deoxy- and 3'-azido-3'-deoxyribonucleoside-5'-triphosphates]. / Ingibirovanie aktivnosti RNK-polimerazy virusa grippa A nekotorymi 3'-amino-3'-dezoksi- i 3'-azido-3'-dezoksiribonukleozid-5'-tri- fosfatami.

The effects of 3'-amino-3'-deoxy- and 3'-azido-3'-deoxyribonucleoside-5'-triphosphates on the RNA synthesis catalyzed by influenza virus A RNA polymerase were studied. All nucleotide analogues tested decreased the RNA synthesis twofold at the inhibitor: substrate ratio about 1:5 (in moles). The hypothetic mechanism of inhibitors action based on the incorporation of inhibitors into the 3'-termini of the RNA chains and subsequent blocking of the RNA chains elongation is proposed. The nucleotide analogues under investigation were several times more effective as compared with the ribavirine 5'-triphosphate, a well-known inhibitor of influenza A virus reproduction.