[Localization of binding sites of E. coli DNA-dependent RNA-polymerase with photosensitive template analogs]. / Lokalizatsiia mest sviazyvaniia DNK-zavisimoi RNK-polimerazy E. coli s fotochuvstvitel'nymi analogami matritsy.

The photoinduced covalent binding of E. coli RNA polymerase with decathymidylic templates containing 5-bromouracil residue has been carried out. Peptides from beta and beta' subunits of the core-enzyme, situated in the DNA-template binding site of the RNA polymerase active center have been localized.

[Preparation and characteristics of monoclonal antibodies against GTP-binding protein from the bovine retina]. / Poluchenie i kharakteristika monoklonal'nykh antitel k GTP-sviazyvaiushchemu belku iz setchatki glaz byka.

Five monoclonal antibodies against the native GTP-binding protein (transducin) from bovine retina have been prepared. By immunoblotting and immunoenzymatic analysis of the isolated alpha- and gamma-subunits of transducin and the beta gamma-subunit complex it was determined that two monoclonal antibodies A3G7 and A3C10 recognize linear antigenic determinants on the alpha-subunit, two other, A3E4 and 3B3, bound specifically to the gamma-subunit, and monoclonal antibodies 1C3 interact only with native transducin. Both antibodies against the alpha-subunit inhibited transducin GTPase activity, whereas antibodies A3E4, 3B3 and 1C3 did not affect it.

[Primary structure of the gamma-subunit of the GTP-binding protein from the bovine retina]. / Pervichnaia struktura gamma-sub''edinitsy GTP-sviazyvaiushchego belka iz setchatki krupnogo rogatogo skota.

The complete amino acid sequence of the gamma-subunit of GTP-binding protein from cattle retina has been established. The polypeptide chain consists of 69 amino acid residues and contains an unusual sequence Cys35-Cys36. The molecular mass of the gamma-subunit is 8008,7.

[Effective method of oligonucleotide-controlled mutagenesis of DNA fragments]. / Effektivnyi metod oligonukleotidnapravlennogo mutageneza fragmentov DNK.

A procedure has been designed for changing specific nucleotides in a DNA sequence with efficiency. The method involves the use of the specially constructed cloning vectors pBRS1, pHS1, and pHS2. These plasmids are derivatives of pBR322 in which the EcoRI-HindIII region has been replaced by synthetic duplexes carrying SmaI, HpaI and XhoI sites, in addition to EcoRI and HindIII sites. The DNA fragment to be mutagenized is cloned in pHS1 (or pBRS1, or pHS2) using restriction sites close to the SmaI and HpaI sites. The recombinant plasmid obtained is digested with one of these enzymes to produce double-stranded DNA with blunt ends. This linear DNA is a substrate for exonuclease III (or T4 DNA polymerase). The digestion under controlled conditions produces duplex with protruding single-stranded 5'-regions which include the site of the desired mutation. The synthesis of DNA by DNA-polymerase I (Klenow's fragment), primed in part by the synthetic oligonucleotide containing the desired mutation, leads to the linear heteroduplex. The closed circular heteroduplex is formed by ligation. After transformation into E. coli, DNA replication generates homoduplexes, some of which contain the mutation. Colony hybridization with the same 32P-labeled oligonucleotide is used to select mutants. The yield of the mutants is 10-20%. This technique can be extended to replicative form of M13 vectors. It can be also applied to any DNA sequence which has a unique site of restriction endonuclease generating blunt ends.

[Analysis of GTP-binding protein from the bovine retina. Complete amino acid sequence of the gamma-subunit]. / Issledovanie GTB-sviazyvaiushchego belka iz setchatki krupnogo rogatogo skota. Polnaia aminokislotnaia posledovatel'nost' gamma-sub''edinitsy.

The complete amino acid sequence of gamma-subunit of GTP-binding protein from bovine retina was determined. The polypeptide chain consists of 69 amino acid residues. It contains unusual sequence -Cys35-Cys36- with disulfide bridge.

[Expression in Escherichia coli cells of mutant human interferon alpha2]. / Ekspressiia v kletkakh Escherichia coli mutantnogo interferona alpha2 cheloveka.

cDNA library was obtained from mRNA isolated from human leukocytes induced by Newcastle disease virus. Clones containing cDNA for alpha 2-interferons were identified by colony hybridization with two synthetic hexadecanucleotides. One of the positive clones contained a NH2-terminal part of cDNA of human interferon identical to cDNA for IFN-alpha 2. The only difference between these two clones was the Ser-8 leads to Asn-8 substitution in deduced sequenced of mature interferons. This mutant interferon, named alpha 2, was expressed in E. coli and its properties were compared with those of interferon alpha 2.

[Conformation NMR analysis of the spatial structure of Buthus eupeus insectotoxin I5A]. / Konformatsionnyi IaMR-analiz prostranstvennoi struktury insektotoksina I5A Buthus eupeus.

1H NMR spectroscopy has been used to collect data related to the spatial structure of insectotoxin I5A Buthus eupeus: pH-dependence of the chemical shifts, deuterium exchange rates of individual amide hydrogens, spin-spin coupling of the H-N-C alpha-H and H-C alpha-C beta-H protons, and nuclear Overhauser effect between distinct protons belonging to amino acid residues remote in the sequence. Molecular conformation in the regions from Asp9 to Cys19 (beta-turn 9-12 and right-hand alpha-helix 12-19) and from Asn23 to Asn34 (antiparallel beta-sheet with the beta-turn 27-30) directly follows from the observed parameters. Pseudoatomic approach of distance geometry algorithm was used to solve the overall folding of the molecule and propose the most probable set of disulfide bridges: Cys2-Cys19, Cys5-Cys31, Cys16-Cys26 and Cys20-Cys33. The spatial structure of insectotoxin I5A B. eupeus demonstrates remarkable similarity with that of a "long" type scorpion neurotoxin V-3 Centruroides sculpturatus.

[Visual rhodopsin. III. Complete amino acid sequence and topography in a membrane]. / Zritel'nyi rodopsin. III. Polnaia aminokislotnaia posledovatel'nost' i topografiia v membrane.

Tryptic hydrolysis of apomembranes, BNPS-skatole cleavage of carboxymethylated rhodopsin and thermolytic digestion of native membranes were carried out to obtain the peptides necessary for the polypeptide chain reconstruction. Gel-filtration on Bio-Gel P-30 in 80% formic acid, ion-exchange and reversed-phase high performance liquid chromatography were used for the peptide isolation. A comparison of rhodopsin hydrophobicity profile with the accessibility of the polypeptide chain in native photoreceptor membranes for proteases allowed to distinguish seven alpha-helical segments and propose a model for arrangement of the protein molecule in the membrane.

[Primary structure of the elongation factor G from Escherichia coli. IX. Structure of peptides generated by cyanogen bromide cleavage of the G-factor isolated on thiol-activated sepharose and of the products of the G-factor cleavage at Asp-Pro bonds. Complete primary structure]. / Pervichnaia struktura faktora élongatsii G iz Escherichia coli. IX. Issledovanie struktury peptidov bromtsianovogo rasshchepleniia G faktora, vydelennykh na tiol-aktivirovannoi sefaroze, i produktov rasshepleniia G-faktora po sviaziam Asp-Pro. Polnaia pervichnaia struktura.

The amino acid sequence of cysteine- and cystine-containing peptides resulting from cleavage of the G-factor by cyanogen bromide has been determined. For structure analysis cyanogen bromide peptides were further degradated using trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease, or limited acid hydrolysis. The products of the G-factor cleavage at Asp-Pro bonds were also studied. The obtained data together with those published earlier permitted to establish the complete primary structure of the elongation factor G. The polypeptide chain consists of 701 amino acid residues and has molecular mass of 77321,46.

[Comparative study of the biological properties of plasmid and natural human interferons]. / Sravnitel'noe izuchenie biologicheskikh svoistv plazmidnykh i prirodnykh chelovecheskikh interferonov.

A comparative study of biological properties of natural and plasmid human interferons was carried out. Natural and leukocyte interferons: alpha (induced by Newcastle disease virus) and gamma (induced by staphylococcal enterotoxin A) as well as natural fibroblastic beta interferon induced by poly(I) X poly(C) were studied in comparison with plasmid interferons alpha-F and alpha-F/D obtained from recombinant bacteria. Antigenic determinants of plasmid interferons alpha-F and alpha-F/D were found to be identical with those of natural and alpha-interferon of man and to differ from those of natural human alpha- and beta-interferons. Both plasmid interferons demonstrated the kinetics of development of the state of resistance to viruses in a human diploid cell culture typical of alpha-interferon but not of gamma-interferon from human leukocytes. Plasmid and natural alpha-interferons have similar anticellular activity for human tumor HeLa cells, similarly activate natural human killer cells and are similarly stabilized in the presence of 0.01 M lantan chloride. All these data permit a conclusion that plasmid human interferons alpha-F and alpha-F/D are analogous and close to the total preparation of natural alpha-interferon from human leukocytes. On the other hand, the range of cells sensitive to the antiviral effect of alpha-F and alpha-F/D interferons is wider than for leukocyte alpha-interferon, and stability on storage and heating is higher.