[Development of a system vector-host in methylotrophic bacteria]. / Razvitie sistemy vektor-khoziain u metilotrofnykh bakterii.

Cloning of methylotropic and other Gram negative bacteria's genes was performed using vectors derived from IncP4 plasmids. Plasmids, such s RSF1010 are 8.8 kb in length, have a high copy number and broad host range and can be mobilized efficiently by a number of conjugative plasmids. IncP4 plasmids have relatively few restriction enzyme's targets suitable for cloning. In this paper the construction of versatile and special purpose IncP4 vectors available for cloning DNA into broad range of bacterial species are described. The seria of versatile vectors involves the transposon containing plasmid and two-replicon vectors. In genetic construction of special vector for direct cloning of restriction fragments the genetic regulation elements of Tn 1 were used. On the base of IncP4 replicon special vectors for construction of bank genes (cosmids) and the vectors for cloning of regulation sequence were also constructed.

[Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida]. / Ekspressiia gena chelovecheskogo interferona alpha F v obligatnom metilotrofe Methylobacillus flagellatum KT i Pseudomonas putida.

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.

[Genome organization of the plasmid of IncQ/P4 group and its vector derivatives]. / Organizatsiia genoma plazmid IncQ/P4-gruppu i vektory, skonstruirovannye na ikh osnove.

The genome organization and functioning of IncQ/P4 plasmids are reviewed. Based on these plasmids, cloning vectors have been constructed for broad host range of gram-negative bacteria. Together with one- and two-replicon vectors for cloning via insertion inactivation of markers, specialized plasmid vectors are described: cosmids, promoter-probe vectors, vectors for direct selection of recombinant molecules. Examples of using broad host range vectors for gene cloning and expression in non-enteric gram-negative bacteria are presented.

[Construction of the gene library of symbiotic nitrogen-fixing Rhizobium lupini]. / Poluchenie biblioteki genov simbioticheskoi azotfiksiruiushchei bakterii Rhizobium lupini.

The gene bank of the symbiotic nitrogen-fixing bacterium Rhizobium lupini (effective strain 359a) was constructed on plasmid pAYC31 that was used to transform Escherichia coli C6000. The bank contains 6600 clones. Restriction analysis showed that the size of the mean insertion fragment in the plasmid in 6.5 kb.

[The effect of amino acids in the asparagine family on the aspartate kinase and homoserine dehydrogenase of ethionine-resistant mutants of Pseudomonas putida]. / Vliianie aminokislot asparaginovogo semeistva na aktivnost' aspartatkinazy i gomoserindegidrogenazy étioninrezistentnykh mutantov Pseudomonas putida.

The activity of aspartate kinase and homoserin dehydrogenase from ethionine resistant mutants Pseudomonas putida 25 and 6 have been studied as affected by amino acids from the family of asparagine. They are characterized by a capacity to the surplus synthesis of methionine. It is shown that mutants have negative regulation of the level of activity of the studied enzymes. It is supposed that the mutations (or mutation) could take place which affected properties of enzymes, which participated directly in the biosynthesis of methionine, in the analogue resistant clones 25 and 6.

[Isolation and properties of genetic engineered human leukocyte interferons alphaI1 and alphaN]. / Vydelenie i svoistva genno-inzhenernykh chelovecheskikh leikotsitarnykh interferonov alphaI1 i alphaN.

Using controlled pore glass chromatography and immunoaffinity chromatography on monoclonal antibodies NK-2 immobilized on Sepharose 4B, the electrophoretically homogeneous interferons alpha N and alpha I1 were isolated from the biomass of gene-engineered Pseudomonas sp. strains. In terms of specific activity on human fibroblast diploid cells, interferon alpha I1 does not differ from interferon alpha A, whereas the specific antiviral activity of interferon alpha N is as low as 2.10(7) JU/mg. The procedures for immunometric assay of interferons alpha N and alpha I1 have been elaborated. Various monoclonal antibodies to interferon alpha A and to natural leucocyte interferon were analyzed; among those the antibodies specifically interacting with interferons alpha N and alpha I1 (but not with interferon alpha A) were identified.