[Functional studies of mutant forms of bacteriophage T7 RNA polymerases containing point substitutions in the motif at the active site of the enzyme].

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.

[Study of the form of bacteriophage T7 RNA polymerase containing point substitutions in the region of functionally important amino acid residues].

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.

[Site-specific mutagenesis of residue Lys-172 of phage T7 RNA polymerase: characterization of transcription properties of mutant proteins].

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.

[Genetic system of selecting point mutations of bacteriophage T7 RNA polymerase].

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.

[Reverse transcriptase of the human immunodeficiency virus: cloning, expression in Escherichia coli, purification of the enzyme, and production of monoclonal antibodies].

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.

[Affinity modification of DNA-dependent RNA-polymerase from phage T7 with 5'-p-fluorosulfonylbenzoyl adenosine: the effect of modification on the interaction with substrates].

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.

[Affinity modification of DNA-dependent RNA-polymerase of phage T7 with 5'-p-fluorosulfonylbenzoyladenosine].

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.

[Extracellular ribonuclease from 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.