[Properties of cloned promoters]. / Izuchenie svoistv klonirovannykh promotorov.

Strong early bacteriophage T7 promoters A2 and A3 and also A2 and lac UV5 promoters with altered segments downstream the initiation of RNA start point were cloned using specially constructed plasmid vectors pBRS188 and PBRS240. The relative signal strengths of these promoters in vivo and in vitro were evaluated and the kinetic parameters of their interaction with RNA polymerase were determined. It has been shown that the nucleotide sequence of the transcribed region plays a significant role in specific promoter-RNA polymerase interaction and that the rate-limiting step of RNA synthesis initiation is different for various promoters.

[Construction of promoter-probe plasmid vector]. / Konstruirovanie plazmidnogo vektora dlia klonirovaniia promotorov.

A new plasmid vector, designated pBRS188 has been constructed for cloning of promoter-containing DNA fragments. This plasmid is a derivative of the E. coli drug-resistance plasmid pBR322 in which a small region (13 base pairs long) within the Tc promoter is eliminated. As a result of the alteration pBRS188 has lost the ability to confer Tc resistance to the host strain. Cloning of foreign DNA fragments, carrying promoters for E. coli RNA polymerase, into the unique EcoRI site of pBRS188 allows to isolate the recombinant TcR transformants. Our construction required the use of new techniques, involving partial hydrolysis of DNA fragments by E. coli DNA polymerase I in the presence of one deoxyribonucleosidetriphosphate and by nuclease S1. An important feature of this method is the ability to regenerate restriction endonuclease recognition sites at junctions of DNA fragments.

[Genetic system of selecting point mutations of bacteriophage T7 RNA polymerase]. / Geneticheskaia sistema otbora tochechnykh mutantov RNK-polimerazy bakteriofaga T7.

Random mutagenesis of the bacteriophage T7 gene 1 was used to delineate the functionally important amino acids residues of its product--T7 RNA polymerase. A two-plasmid system has been constructed for the phenotypic selection of the mutants. Nine mutants were isolated; the RNA polymerase with Tyr-639----Asp substitution has been shown to be completely inactive. Methods were developed for the rapid purification of plasmid-encoded mutant proteins, facilitating their future biochemical analysis.

[Affinity modification of DNA-dependent RNA-polymerase of phage T7 with 5'-p-fluorosulfonylbenzoyladenosine]. / Affinnaia modifikatsiia DNK-zavisimoi RNK-polimerazy faga T7 5'-p-ftorsul'fonilbenzoiladenozinnom.

The affinity modification of the DNA-dependent RNA-polymerase of bacteriophage T7 was carried out by using the specific irreversible inhibitor, 5'-p-fluorosulfonylbenzoyladenosine. The inhibitor was found to bind to the enzyme's active site; the kinetic constants of the modification were calculated. The stoichiometry of the covalent E.I-complex formed was determined by using the 14C-labeled inhibitor.

[Site-specific mutagenesis of residue Lys-172 of phage T7 RNA polymerase: characterization of transcription properties of mutant proteins]. / Sait-spetsificheskii mutagenez ostatka Lys-172 RNK-polimerazy faga T7: kharakterizatsiia transkriptsionnykh svoistv mutantnykh belkov.

Lys-172 residue of bacteriophage T7 RNA polymerase (T7RP) was substituted for Leu and Gly and Lys-172, Arg-173 were deleted by the site-directed mutagenesis using synthetic oligonucleotides. The specific activity of all mutant enzymes did not differ significantly from that of the wild-type (w.t.) T7RP while for Gly-172 mutant (G172) it was somewhat lower. Leu-172 (L172) and deletion (DEL172-3) mutants were able to direct RNA synthesis on the templates lacking the T7 promoter. DEL172-3 was not able to synthesize extraneous RNA sequences in addition to the expected run-off transcripts. L172 and DEL172-3 mutants revealed altered template specificity toward various DNA templates and showed the lower stability of enzyme-promoter complexes. The possible role of Lys-172 likely belonging to an interdomain "stretch" is discussed.

[Study of the form of bacteriophage T7 RNA polymerase containing point substitutions in the region of functionally important amino acid residues]. / Issledovaniia forma RNK-polimerazy bakteriofaga T7, soderzhashchikh tochechnye zameny v oblasti funktsional'no bazhnykh aminokislotnykh ostatkov.

A study was carried out on the influence of point mutations of the functional amino acid residues on the secondary and ternary structure of bacteriophage T7 RNA polymerase as well as on the activity of the enzyme. A change in residue 631 is accompanied by significant changes in secondary structure (alpha-->beta transition). The substitution Lys-172 Leu changes both the secondary and ternary structures whereas the deletion of residues 172-173 does not lead to such changes. Changes in residues 631-632 do not affect the ability of the enzyme to bind the promoter and/or the synthesis of a full-length transcript but disturbs phosphodiester bond formation.

[Affinity modification of DNA-dependent RNA-polymerase from phage T7 with 5'-p-fluorosulfonylbenzoyl adenosine: the effect of modification on the interaction with substrates]. / Affinnaia modifikatsiia DNK-zavisimoi RNK-polimerazy faga T7 5'-p-ftorsul'fonilbenzoiladenozinom: vliianie modifikatsii na vzaimodeistvie s substratami.

T7 RNA polymerase, covalently modified with 5'-p-fluorosulfonylbenzoyl adenosine, looses the ability of binding the promoter (pGEM-2 plasmid) and poly(dC) template as well as the initiating nucleoside triphosphate (GTP). However the enzyme retains the unspecific binding with DNA fragments of considerable length.

[Reverse transcriptase of the human immunodeficiency virus: cloning, expression in Escherichia coli, purification of the enzyme, and production of monoclonal antibodies]. / Obratnaia transkriptaza virusa immunodefitsita cheloveka: klonirovanie, ékspressiia v Escherichia coli, ochistka fermenta i poluchenie monoklonal'nykh antitel.

To express HIV-1 reverse transcriptase in E. coli a number of genetic constructions containing reverse transcriptase and virus protease nucleotide sequences was obtained. The products of expression were characterized; monoclonal antibodies to reverse transcriptase were produced. The purification of reverse transcriptase was carried out. The substantial proteolysis of reverse transcriptase during purification was shown. The purified preparation is predominantly, an active protein with Mr 57 kDa. Some properties of this protein differed from the reverse transcriptase isolated from HIV.

[Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene]. / Ispol'zovanie gena supressornoi tRNK dlia ispravleniia nonsens-mutatsii v gene distrofina.

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.

[Directed mutagenesis of chromosomal genes of Escherichia coli in vivo: construction of RecA- and HtpR- mutants]. / Napravlennyi mutagenez khromosomnykh genov Escherichia coli in vivo: konstruirovanie RecA- i HtpR- mutantov.

The deletions in Escherichia coli chromosomal genes recA and htpR were constructed using the site-directed mutagenesis techniques. The obtained RecA- mutants are UV-sensitive and have a phenotype defective for the homologous DNA recombination. HtpR- mutant is temperature sensitive for growth and deficient in intracellular proteolysis. As a result a HtpR- mutant seems to be a preferable candidate for attempting to synthesize efficiently any alien protein in Escherichia coli cells.

[Recovery of activity of the gene coding for tetracycline resistance in the plasmin pBRS188]. / Vosstanovlenie aktivnosti gena ustoichivosti k tetratsiklinu plazmidypBRS188.

The spontaneous recovery of activity of tet gene deleted of the promoter region was studied. Plasmid pBRS188 was used as a model for studying this problem. The plasmid has the fragment of tet gene of pBR322, from which it originates, between the sites of restriction endonucleases EcoRI and HindIII cleavage resulting in inactivation of tet promoter. E. coli cells harbouring the plasmid were shown to revert the TcR phenotype with the frequency 10(-9). The gene activation coincided with intraplasmid recombination revealed by restriction analysis. In some cases the recovery of tet gene activity coincided with the formation of multimeric plasmids.

[Detection of the provirus of the bovine leukemia virus in bovine peripheral blood leukocytes by means of DNA hybridization]. / Obnaruzhenie provirusa virusa leikoza krupnogo rogatogo skota v leikotsitakh perifericheskoi krovi krupnogo rogatogo skota metodom DNK-gibridizatsii.

The method allowing the detection of BLV proviral DNA in the peripheral blood leukocytes of cattle is reported. Cell DNA from leukocytes used without preliminary cultivation was dot-hydridized with 32P-labeled plasmid that included a fragment of BLV proviral DNA. In parallel, sera from the cattle under study were tested by immunodiffusion assay (ID). The results indicate that dot-hybridization assay is more sensitive as a diagnostic test than ID because it detects BLV infection in apparently normal cattle which was seronegative by ID.

Assay of aminoglycoside phosphotransferase in situ.

A method for the evaluation of the aminoglycoside phosphotransferase activity in bacterial colonies directly is described. The method is based on the ability of the enzyme to modify the substrate immobilized on carboxymethylcellulose paper. The sensitivity and accuracy of the method were tested by comparing the results of the present assay to those obtained with conventional procedures. The method seems to be particularly useful for the detection within a bacterial population producing aminoglycoside phosphotransferase of those cells which do not make the enzyme and for rapid determination of the relative levels of enzyme produced by different clones.

[Extracellular ribonuclease from Bacillus thuringiensis]. / Vnekletochnaia ribonukleaza iz Bacillus thuringiensis.

The ability of the strain Bacillus thuringiensis var. subtoxicus to produce extracellular ribonuclease (ribonuclease Bt) was studied. It was found that the culture medium possesses a RNA-depolymerizing activity whose maximum is observed 4-5 hours after the beginning of the linear growth phase. A three-step chromatography of the culture extract on phosphocellulose resulted in a homogeneous enzyme with a molecular mass of 12000 Da. The enzyme showed the maximum activity towards RNA at pH 8.5, catalyzed the hydrolysis of polyribonucleotides and guanosine-2',3'-cyclophosphate. Hence, the enzyme can be related to base-nonspecific cyclizing ribonucleases showing the guanylic specificity towards nucleoside-2',3'-cyclophosphates.

[Stability of human immunodeficiency virus to azidothymidine. II. Kinetic characteristics of "AZT-resistant" mutant forms of reverse transcriptase]. / Issledovaniia ustoichivosti virusa immunodefitsita cheloveka k azidotimidinu. II. Kineticheskie kharakteristiki "AZT-ustoichivykh" mutantnykh form obratnoi transkriptazy.

Prolonged treatment of AIDS patients with azidothymidine results in the development of resistance to the drug which correlates with the appearance of point mutations in the reverse transcriptase (RT) coding region within the HIV-1 pol gene. Kinetic studies of interactions of wild type RT and its mutants harbouring the above mutations with substrates and azidothymidine 5'-triphosphate (AZTTP) have been carried out. The complete mutant containing all the above described mutations possess the highest resistance on all the templates tested. Significant increases in resistance for mutants 67,70,215 and 67,215 on all the templates have also been observed. Inhibition of mutant enzymes by AZTTP depends on the template used.

[Functional studies of mutant forms of bacteriophage T7 RNA polymerases containing point substitutions in the motif at the active site of the enzyme]. / Funktsional'nye issledovaniia mutantnykh form RNK-polimerazy bakteriofaga T7, soderzhashchikh tochechnye zameny v motive v aktivnogo tsentra fermenta.

Monomeric DNA and RNA polymerases contain a number of conserved motifs. By means of random and oligonucleotide-directed mutagenesis the point mutants of bacteriophage T7 RNA polymerase containing the amino acid substitutions in motif B which is the part of the active site were obtained. The kinetic properties of the mutants were studied. The results obtained suggest that amino acid residues 636, 639 and 646 are involved in the binding of the initiating GTP, the selection of correct nucleoside triphosphate and interaction with the promoter, respectively.

[Studies of the resistance of the human immunodeficiency virus to azidothymidine. I. Kinetic studies of the interaction between reverse transcriptase and a series of its mutant forms with substrates and azidothymidine-5'-trisphosphate]. / Issledovaniia ustoichivosti virusa immunodefitsita cheloveka k azidotimidinu. I. Kineticheskie issledovaniia vzaimodeistviia obratnoi transkriptazy i riada ee mutantnykh form s substratami i azidotimidin-5'-trifosfatom.

Prolonged therapy of AIDS patients with azidothymidine results in the development of resistance to the drug. This phenomenon is accompanied by the appearance of point mutations in the pol gene coding for reverse transcriptase (RT). Kinetic studies of interactions of wild type RT and its forms containing the above-mentioned mutations with substrates and azidothymidine 5'-triphosphate have been carried out. Considerable differences in the affinities of RT and the mutants for RNA and DNA heteropolymer templates were established. The mutations did not affect the RT affinity for dNTP; however, its location influenced considerably the inhibition of the reaction with azidothymidine 5'-triphosphate.