[Study of antigenic structure of HIV-1 protein p24 using monoclonal antibodies]. / Izuchenie antigennoi struktury belka p24 VICh-1 s pomoshch'iu monoklonal'nykh antitel.

During the experiments 4 murine and 3 rat hybridomas producing monoclonal antibodies (MAb) against the protein p24 of human immunodeficiency virus type 1 (HIV-1) have been obtained. Using the immunoblotting technique, it was established that all the species of MAb reacted with the same viral proteins which are derivatives of gag gene--p24 and p55. The properties of MAb have been studied in competitive binding. Their ability of binding to different fragments of the gag protein produced by the recombinant plasmids in E. coli cells have been investigated in ELISA. The analysis of the findings suggests that the HIV-1 protein p24 contains at least 3 antigenic epitopes. All species of MAb reacted with 3 different HIV-1 strains and 2 HIV-1 isolates, but failed with 2 different HIV-2 strains. The only MAb NS5E4 can be used as an immunosorbent in the antigenic capture reaction.

[Introduction of aminoalkyl groups into prepared synthetic oligodeoxyribonucleotides by transamination of cytosine residues]. / Vvedenie aminoalkil'nykh grupp v gotovye sinteticheskie oligodezoksiribonukleotidy putem pereaminirovaniia ostatkov tsitozina.

The bisulphite-catalysed transamination of cytosine residues by means of ethylenediamine generally used for the natural nucleic acids modification has been extended on relatively short synthetic oligonucleotides. One of the aminoalkyloligonucleotides thus obtained has been used for preparing a biotinylated hybridisation probe.

[Directed reconstruction of the influenza virus hemagglutinin gene]. / Napravlennaia rekonstruktsiia gena gemaggliutinina virusa grippa.

A new approach to create chimeric genes by directed exchange of oligonucleotide fragments was developed. By oligonucleotide-directed mutagenesis a few deletion mutants of the influenza virus hemagglutinin (HA) gene were obtained. These variants of HA gene contain unique restriction sites in DNA regions coding for the A and B epitopes of the HA molecule. The obtained special vectors may be used for cloning DNA fragments coding for new amino acid sequences in internal sites of the HA gene.

[Evaluation of the relative effectiveness of hybridization probes prepared by action of hydrazides of biotin derivatives on DNA]. / Otsenka otnositel'noi éffektivnosti gibridizatsionnykh zondov, poluchennykh deistviem gidrazidov proizvodnykh biotina na DNK.

Efficiencies of biotinylated DNAs as hybridisation probes in a model system of non-radioactive detection were compared. Probes were obtained by interaction of single-stranded DNA and each of four different hydrazides of biotin derivatives. The most sensitivity in detection of complementary target was obtained using (biocytin hydrazide)-treated DNA. Relations between hydrazide structures and sensitivity of biotinylated probes are discussed.

[Construction and expression in Escherichia coli of a gene of hybrid hemagglutinin H1-H3 of influenza virus]. / Konstruirovanie i ékspressiia v Escherichia coli gena gibridnogo gemaggliutinina podtipa H1-H3 virusa grippa.

The hybrid gene of influenza virus hemagglutinin (HA) of the H1-subtype, carrying the sequence coding for the fragment of H3-subtype antigenic site B, was constructed. The product of expression of this gene in E. coli was obtained as a fusion protein with beta-galactosidase. The chimeric protein was shown to retain the antigenic properties of HA of H1-subtype and to interact specifically with antibodies against the synthetic peptide corresponding to the B site fragment of HA of the H3-subtype.

[Cloning and expression of the hemagglutinin gene of influenza virus subtype H1 in Escherichia coli]. / Klonirovanie i ékspressiia v Escherichia coli gena gemaggliutinina virusa podtipa H1.

The full-length copy of the hemagglutinin gene of influenza virus was inserted into M13 phage DNA. The DNA sequence coding for the hydrophobic prepeptide was removed from the gene by oligonucleotide-directed mutagenesis. The possibilities of expression of the full-length and mutant genes in E. coli were investigated. The beta-galactosidase-hemagglutinin fusion proteins were isolated. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess of viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.

[Mutagenesis directed by phosphotriester analogs of oligonucleotides: a way to site-specific mutagenesis in vivo]. / Mutagenez, napravlennyi fosfotriéfirnymi analogami oligonukleotidov, - put' k sait-spetsificheskomu mutagenezu in vivo.

A new approach is proposed to obtain the directed mutations in the gene under study. The technique is based on using alkylphosphotriester analogues of oligodeoxyribonucleotides as site-specific mutagens. The deletion C in lacZ' gene of bacteriophage M13mpB was obtained by cotransfection of Escherichia coli cells with a mix of DNA and phosphotriester analogues of oligonucleotides.

[Mutagenic properties of phosphotriester analogs of oligodeoxyribonucleotides]. / Mutagennye svoistva fosfotriéfirnykh analogov oligodezoksiribonukleidov.

The high effectivity of using phosphotriester analogs of oligonucleotides for aimed mutagenesis in vitro and in vivo was shown. A general scheme, describing the mutagenic effects of phosphotriester analogs of oligonucleotides and their natural homologs, was derived by analysis of data on the structures of the obtained mutants. This scheme can serve as a foundation for selecting the structure of effective agents for aimed mutagenesis.

[Effectiveness of substituting E. coli DNA-polymerase for DNA-polymerase A in oligonucleotide-directed mutagenesis]. / Effektivnost' zameny DNK-polimerazy A DNK-polimerazoi I E. coli v oligonukleotidnapravlennom mutageneze.

The possibility to use the E. coli intact DNA polymerase I in the oligonucleotide-directed site-specific mutagenesis of DNA has been studied. Optimal conditions of the extension activity of this enzyme were found. We have shown that the substitution of the Klenow fragment of the E. coli DNA polymerase by the intact DNA polymerase I did not decrease the efficiency and fidelity of the oligonucleotide-directed mutagenesis.

[Variants of phage M13 DNA containing a fragment of the beta-galactosidase gene--a convenient mutation system for the study of oligonucleotide-directed mutagenesis]. / Proizvodnye DNK faga M13, soderzhashchie fragment gena beta-galaktozidazy--udobnaia mutatsionnaia sistema dlia issledovaniia napravlennogo oligonukleotidami mutageneza.

A model system is developed to test oligonucleotide-directed mutations: T----C transition, T and C deletions (delta T and delta C), C insertion, double mutations (A----G, delta T), (T----C, A----G), and large oligonucleotide deletions (36 or 44 nucleotides). The system includes 9 variants of the phage M13 DNA carrying fragment of beta-galactosidase gene, and oligodeoxyribonucleotides partially noncomplementary to DNA sequence of this gene. Six variants are obtained by the site-localized mutagenesis, the other were described earlier. Induced mutations are easily tested by phenotype change of transformed bacteria (Lac+----Lac-); by formation or loss of the sites for BamHI and EcoRI restrictases; by DNA hybridization with 32P-labeled oligonucleotides; and by DNA sequencing by the Sanger method. The system is used to study the role of some factors, such as completeness of RF DNA synthesis, thermal stability of the oligonucleotide: DNA complex, quality of enzymes and substrates used in polymerase reaction, mutation type or the efficiency of mutagenesis. A number of unexpected mutations were observed in the course of oligonucleotide-directed mutagenesis. Lower yields of some mutants induced by oligonucleotides are shown to be due to the action of repair systems of bacteria.

[Preparation of fluorescent-labeled DNA and its use as a probe in molecular hybridization]. / Poluchenie fluorestsentno mechennoi DNK i ispol'zovanie ee v kachestve zonda pri molekuliarnoi gibridizatsii.

A method of the fluorescent-labeled DNA preparation for visualization of the complementary nucleotide sequences has been developed. Polynucleotide probes were alkylated randomly by 4-(N-methylamino-N-2-chloroethyl)-benzylamine followed by modification with such fluorochromes as dansyl chloride or fluorescein isothiocyanate (FITC). It was found that the FITC but not dansyl-labeled polynucleotides could serve as efficient probes when about 4% of nitrogen bases were modified. The conditions minimizing the loss of the alkylated bases from DNA were determined. The procedure for hybridization with FITC-labeled DNA as a probe is described, concentration of DNA probe being about 4 ng/mm2 of the nitrocellulose filter.

[Site-localized mutagenesis directed by phosphotriester analogs of oligonucleotides]. / Sait-lokalizovannyi mutagenez, napravliaemyi fosfotriéfirnymi analogami oligonukleotidov.

17- and 20-mer oligodeoxyribonucleotides and their analogues, containing one to four phosphate groups esterified with ethyl alcohol in different positions of oligonucleotide chain, were synthesized by modified triester method. Ethylated di- and trinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. The structures of the oligonucleotides were confirmed by Maxam-Gilbert sequencing method. Oligonucleotides were not totally complementary to the N-terminal region of lac Z'gene (coding for N-terminal fragment of beta-galactosidase) of phage M13mpB DNA and induced the formation of the proposed deletion mutant DNA M13mp1 delta T. Phosphotriester analogues were more effective mutagens as compared to phosphodiester oligonucleotides due to their stability to nucleases. The use of E. coli DNA-polymerase I provided the increase in the mutant yields in case of the phosphotriester analogues. The stability of the analogues to 5'----3'----5'-endonuclease action, the specificity of oligonucleotide: DNA binding and the structure of mutant DNA were studied by the Sanger sequencing method.

[Mutagenesis directed by phosphotriester analogs of oligonucleotides]. / Mutagenez, napravlennyi fosfotriéfirnymi analogami oligonukleotidov.

20-mer oligodeoxyribonucleotides d-ACGACGG (R') CCAG (R'') TGATCCGTA, where R' = R'' = H (20), F' = Et, R'' = H (20-Et), or R' = R'' = Et (20-Et2) were synthesized by modified triester method. Ethylated dinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. Structures of oligonucleotides were confirmed by Maxam - Gilbert method. Mutagenesis induced by oligonucleotides was studied on DNA of M13mpB phage. Oligonucleotides were not totally complementary to this DNA in the region of 4-11 codons of Z'-gene. They all were shown to direct the formation of the designed deletion mutants, phosphotriester analogues (20-Et) and (20-Et2) being more effective mutagens. The specificity of oligonucleotides: DNA binding and mutant DNA structure were shown by Sanger method.

[Specific modification of DNA at E. coli RNA-polymerase binding sites]. / Spetsificheskaia modifikatsiia DNK v mestakhh sviazyvaniia RNK-polimerazy E. coli.

Specific modification of promoter regions of DNA has been studied. Plasmid pK56B1 DNA has been used as a model to test RNA-polymerase binding with DNA under various conditions. RNA-polymerase is shown to form specific complexes with DNA which are stable in solutions with a moderate ionic strength (0.1-0.2 M NaCl), under pH 5-8 in the presence of 0.5 M O-methylhydroxylamine of O-delta-aminooxybutylhydroxylamine. Escherichia coli JM103 cells have been transfected with DNAs treated with 0.5 M O-methylhydroxylamine at 37 degrees C, pH 5.2. The inactivation effects of the mutagen on single-stranded DNA of bacteriophage M13 m p1, double-stranded form of this bacteriophage (replicative form-RF) and on the complex of RNA-polymerase with RF DNA have been compared. The obtained data confirmed the specificity of reagent action with DNA sites binding with the enzyme. Selectivity of promoters modification has been confirmed also by the analysis of M13 m p1 DNA mutations induced in lacZ' gene by delta-aminooxybutylhydroxylamine effect on the DNA complex with DNA-polymerase.