[The characterization of Francisella tularensis mutants and subspecies by using monoclonal antibodies]. / Kharakteristika mutantov i podvidov Francisella tularensis s pomoshch'iu monoklonal'nykh antitel.

Monoclonal antibodies to F.tularensis cells, subspecies holarctica, were studied for the capacity of reacting with F.tularensis, subspecies nearctica, and its mutants having lower virulence and altered capacity for inducing protective immunity to tularemia in laboratory animals. Among the antibody-producing hybridoma clones under study, clones F8/67 and C7/65 capable of distinguishing the mutants of F.tularensis, subspecies nearctica, with lower virulence than that of the initial strain were selected. Antibodies of these hybridoma clones did not interact with the antigens of the initial virulent strains of F.tularensis, subspecies nearctica, while giving pronounced reaction with the antigens of its mutants. Close F8/67 produced IgG antibodies and clone C7/65, IgM antibodies. As shown in immunoblotting, antibodies produced by these hybridoma clones bound with proteins of F.tularensis cell membranes.

[Mutations in Francisella tularensis, decreasing the virulence of these bacteria and leading to a change in the immune response upon infection of experimental animals with them]. / Mutatsii u Francisella tularensis, snizhaiushchie virulentnost' étikh bakterii i privodiashchie k izmeneniiu immunnogo otveta pri zarazhenii imi éksperimental'nykh zhivotnykh.

The research was aimed at isolation of Francisella tularensis mutants possessing the decreased virulence for experimental animals and mediating the changes in the animal immune response. A number of spontaneous and induced mutants of the American and European subtypes of Francisella tularensis were selected for antibiotics resistance or detergent sensitivity. All the obtained mutants have the decreased virulence and differ in their ability to induce the protective antitularemia immunity or ability to induce the humoral immune response in the laboratory animals. The dimeric immunoprecipitation in gel as well as immunoblotting have shown the mutations decreasing the virulence to cause the loss by bacteria of a number of antigenic structures (in case the virulence is completely lost) or changes in antigenic structure resulting in inability of bacteria to induce the humoral immune response when immunizing the laboratory animals. The latter occurs in partially virulent mutants of the vaccine mutant type. The concomitant changes in virulence, ability to cause protective immunity or humoral immune response of the mutants is discussed.

[The genetic transfer, heredity and phenotypic expression of plasmid RP4::Mu cts genes in Bacillus cereus]. / Geneticheskii perenos, nasledovanie i fenotipicheskoe vyrazhenie genov plazmidy RP4::Mu cts v Bacillus cereus.

The transcipients were obtained in intrageneric matings of E.coli donor harbouring the plasmid PR4::Mu cts 62 with Bac. cereus GP7 recipient cells with the frequency 10(-9). The transcipient clone Bac. cereus 682 was selected having stably inherited and expressed the hybrid plasmid PR4::Mu cts 62 genes for antibiotic resistance and temperature sensitivity. Production of the bacteriophage Mu cts 62 particles was not registered in the bacillary transcipient cells. The plasmid RP4::Mu cts 62 genes were localized in the chromosome of Bac. cereus 682 transcipient by the blot-hybridization technique with 32P-labelled DNA of the bacteriophage Mu cts 62 and the plasmid PR4. The transcipient of Bac. cereus 682 has the donor properties and transfers the RP4::Mu cts 62 genes to recipient cells of Bac. cereus DSM 318 with the frequency of 10(-6)-10(-7). The expression and transfer of the gram-negative plasmid genes in gram-positive bacterial cells are discussed.

[Construction and analysis of the tularemia pathogen gene library in Escherichia coli]. / Sozdanie i analiz biblioteki genov vozbuditelia tuliaremii v kishechnoi palochke.

The library of tularemia causative agent genes cloned on the pHC79 plasmid and the partial clonotek of these agents genes in Escherichia coli cells have been constructed. The immunochemical analysis has revealed seven clones of Escherichia coli harbouring the recombinant plasmids and expressing francisella antigens. The cloned sequences of francisella DNA as well as the recombinant plasmids containing them and coding for francisella antigens are capable of specific hybridization with the DNA from Francisella tularensis strains and Francisella novicida strain U112. The cloned DNA sequences have the properties of the genetic radiospecific molecular DNA probe.

[Functional properties of the pOV13 plasmid as a vector for DNA cloning in a broad spectrum of gram negative bacteria]. / Funktsional'nye svoistva plazmidy pOV13 kak vektora dlia klonirovaniia DNK v shirokom kruge gramotritsatel'nykh bakterii.

The birepliconed plasmid pOV13 possesses all the properties of a vector for DNA cloning in a broad host range of bacterial cells. pOV13 is transfered by transformation and stably inherited by Escherichia coli, Brucella, Pseudomonas cells determining the resistance to streptomycin, tetrocycline and kanamycin in these bacteria. The plasmid pOV13 is a multicopy plasmid optimal in replication capacity (23kb). The plasmid carries single sites for some restriction endonucleases that are used for DNA cloning, including some restriction sites in antibiotic resistance genes. The examples of DNA cloning with the selection of recombinant clones by the insertional inactivation of kanamycin or tetracycline resistance and expression of the cloned DNAs are presented.

[Isolation and characteristics of a recombinant pOV13 plasmid as a vector for DNA cloning in a broad range of bacterial hosts]. / Poluchenie i kharakteristika rekombinantnoi plazmidy pOV13 kak vektora dlia klonirovaniia DNK v shirokom kruge bakterial'nykh khoziaev.

The hybrid plasmid pOV13 proposed as a potential vector for DNA cloning in a broad bacterial host range has been constructed on the basis of the broad host range plasmid RSF1010 and a shortened derivative of RP4, the plasmid pVZ115 serving a marker DNA fragment. The plasmid pOV13 contains the genes for streptomycin, kanamycin and tetracycline resistance and single cleavage sites for restriction endonucleases BamHI, BgIII, SalI, SmaI, PvuII, XhoI, as well as double cleavage sites for restriction endonucleases PstI and HindIII permitting one to clone DNA with insertional inactivation of genes. The physicogenetical map of the birepliconed plasmid pOV13 is presented.

[Use of the DNA-DNA hybridization method on filters for determining the toxic potency of atypical strains of Vibrio cholerae isolated from natural sources]. / Ispol'zovanie metoda DNK-DNK-gibridizatsii na fil'trakh dlia opredeleniia toksigennoi potentsii atipichnykh shtammov Vibrio cholerae, vydelennykh iz prirodnykh istochnikov.

The methods of the radioimmunoassay and the blot hybridization of restricted fragments of chromosomal DNA have been used for the characterization of V. cholerae atypical strains isolated from the natural environment. For all strains under study, the radioimmunoassay has been found to yield the most sharply defined data characterizing their atoxigenicity. The absence of the structural genes of toxin in the chromosomes of these strains has been shown by the method of blot hybridization. Some methodological simplifications of blot hybridization, having no adverse effect on the sensitivity of this method, have been tested.

[Localization of structural genes of Vibrio cholerae cholerae toxin using the recombinant plasmid RP4omega elt]. / Lokalizatsiia strukturnykh genov kholernogo toksina Vibrio cholerae cholerae s ispol'zovaniem rekombinantnoi plazmidy RP4omega elt.

The recombinant plasmid RP4 omega elt carrying Escherichia coli heat-labile enterotoxin elt genes with 70-80% homology with genes vct of Vibrio cholerae has been constructed. We used this plasmid to determine localization of the cholerae toxin genes vct on the map of Vibrio cholerae cholerae. Two types of the donors were revealed in matings of 10 strains of V. cholerae cholerae 569B/RP4 omega elt with the polyauxotrophic recipients RV31 and RV175: some strains had enhanced frequency of mobilization of ilv-1 and lys-6 markers, the others--of trp-1. Our data suggest that structural vct genes are located within two regions of V. cholerae cholerae 569B chromosome: trp-1 and ilv-1--lys-6.

[Properties of the plasmid pFT15/10-1 isolated from the vaccine strain of Francisella tularensis]. / Svoistva plazmidy pFT15/10-1, vydelennoi iz vaktsinnogo shtamma Francisella tularensis.

Plasmid, designated pFT15/10-1, was isolated from Francisella tularensis vaccine strain 15/10. The plasmid is presented by the homogeneous 5.02 +/- 0.054 Md monomeric circular DNA molecules in electron microscopic preparations. Plasmid size is 7-7.3 kb as defined by electrophoresis in agarose gel. The restriction analysis has revealed that plasmid pFT15/10-1 possesses a single specific cleavage site for restriction endonuclease EcoRI, two sites for restriction endonucleases BamHI, BgIII, HincII, HindIII, PstI, three sites for BglI and SalI, some for AluI, TagI, MvaI, CfrI. Plasmid is not digested by restriction endonucleases SmaI, XmaI, KpnI, MluI. Restriction map of the plasmid was constructed for most frequently used restriction endonucleases.

[Analysis of insertions of the determinant of drug resistance to kanamycin and chloramphenicol into bacteriophage lambda att80]. / Analiz vnedrenii determinant lekarstvennoi ustoichivosti k kanamitsinu i khloramfenikolu v bakteriofag lambda att80.

The insertion sites of elements Tn9 and Tn601 which determine chloramphenicol and kanamycin resistance have been detected restriction analysis. The functioning of transposons i.e. their stability or instability, has been found to influence the specificity of their insertions into the genome of lambda att80 bacteriophage. During transposition from stable integration sites both transposons are inserted into the regions of the lambda att80 bacteriophage genome, definite for each transposon. However, during transposition from the site of unstable integration both determinants of drug resistance are inserted into different regions of the phage genome.

[Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location]. / Spetsifichnost' vnedreniia Tn9 v genom bakteriofaga liambda att80 v zavisimosti ot ego predshestvuiushchei lokalizatsii.

It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA. The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability. When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb. In the course of transposition of Tn9 from chromosomal attTn9N site the transposon inserted into the lambda att80 site with coordinates 26,5 +/- 5 kb. In the latter case, the increase of Tn9 single-stranded loop and the appearance of two new HindIII cleavage sites were observed in heteroduplex experiments. The data were interpreted as indicating structural rearrangements of Tn9 or linked sequences in the course of transposition.

[Role of Escherichia coli K-12 lambda phage receptors in transfection and plasmid transformation]. / Rol' retseptorov faga lambda u kletok Escherichia coli K-12 v transfektsii i plazmidnoi transformatsii.

The dependence of competence of Escherichia coli cells in transfection and plasmid transformation from phage lambda receptor protein was studied. Mal+ variants, sensitive to phage lambda (Mal+ lambda S) were obtained from strains with impaired lambda-phage receptor protein (Mal-lambda R). Maximum increase of transfection (4.7-fold) was observed in JC411Mal+ lambda S strain. The efficiency of plasmid transformation of pMB9 and RP4 DNAs was also higher in the strain with functioning lambda-receptor protein.

[Mechanism of plasmid ColEl and pMB-9 mobilization by plasmid F'lac+ in Escherichia coli K-12]. / Izuchenie mekhanizma mobilizatsii plazmid ColEl i pMB-9 plazmidoi F'lac+ u Escherichia coli.

The genetic control and mechanism of mobilization of the non-conjugative plasmids ColE1 and pMB-9 by the conjugative plasmids was orived to be recA-independent process in contrast to the mobilization of the chromosomal marker pro. Acridine orange and ethidium bromide curing data together with the results of electrophoretic analysis of plasmid DNA suggest that the plasmids F' lac+ and pMB-9 as well as F' lac+ and ColE1 remain autonomous after their contransfer to recipient cells. These data argue in favour of non-recombinational nature of the plasmid mobilization process. The possibility of transmission of a non-conjugative plasmid without transmission of a conjugative one from the donor strain carrying both plasmids was established. The results obtained are discussed with respect to the hypothesis on the effect of diffusible products encoded by the conjugative plasmid and required of the mobilization of the non-conjugative plasmid.