[Cloning and insertion mutagenesis of DNA fragment coding for the luminescent system of Photobacterium leiognathi]. / Klonirovanie i insertsionnyi mutagenez fragmenta DNK, kodiruiushchego liuminestsentnuiu sistemu Photobacterium leiognathi.

Fragments of DNA, obtained from the luminescent bacterium Photobacterium leiognathi and inserted into the plasmid pBR322, were found to code for the luminescence expressed in E. coli cells. The genetic functions necessary for light production in E. coli are localized on a DNA fragment of about 7 kbp. The insertion mutagenesis was used to define the luminescence functions encoded by the hybrid plasmid.

[Recombination of plasmids with inverted repeats in Bacillus subtilis cells]. / Rekombinatsiia plazmid s invertirovannymi povtorami v kletkakh Bacillus subtilis.

The plasmid pNB48 carring a pair of genetically distinct inverted repeats is constructed on the basis of plasmid pSM19035 replicon. The single Campbell-type recombination between inverted repeats of two circular monomers leads to the formation of the so called "inverted" dimers in Bacillus subtilis cells. The gene conversion is observed in the course of this recombination. The mechanism of structural rearrangements of plasmids with inverted repeats that was postulated earlier is confirmed now. Some replicon features may influence the recombination of plasmids in Bacillus subtilis cells. The plasmid pNB48 may be a suitable model for studying the genetic recombination in Bacillus subtilis.

[Plasmid inverted repeats provide for duplication or precise excision of DNA inserts]. / Plazmidnye invertirovannye povtory obespechivaiut duplikatsiiu ili tochnoe vyrezanie vstroennykh v nikh fragmentov DNK.

A plasmid containing inverted repeats is constructed in Bacillus subtilis. Insertion of DNA fragments into the plasmid inverted repeats results either in the precise excision of the insert or in its duplication in the opposite inverted repeat. These rearrangements are due to the presence of inverted repeats only. Two recombination events are possibly responsible for these phenomena. During the first step of the recombination two plasmid monomers form a dimer molecule. During the second step the intramolecular recombination between the direct repeats in the dimer structure leads to the formation of two rearranged plasmid monomers devoid of insertion or containing two DNA inserts. Proposed dimeric intermediate is unstable in B. subtilis. However, it is isolated from Escherichia coli recA, cells. Plasmids containing the inverted repeats can serve as a model to study plasmid recombination in B. subtilis cells.

[Cloning of the genes controlling the biosynthesis of bacitracin in Bacillus licheniformis]. / Klonirovanie genov, kontroliruiushchikh biosintez batsitratsina u Bacillus licheniformis.

The DNA fragment from bacitracin-producing Bacillus licheniformis strain is cloned on pMX39 vector plasmid in Bacillus subtilis cells. Bacillus subtilis cells carrying the cloned fragment inhibit the growth of bacitracin-sensitive tester strain. The observed inhibition of growth is due to the production by Bacillus subtilis of bacteriocin substance that is identified as bacitracin by TLC-chromatography. In contrast to the data published earlier it is shown that Bacillus subtilis can in fact produce the small amounts of bacitracin. Introduction of the cloned Bacillus licheniformis DNA into Bacillus subtilis cells stimulates this bacitracin production. The restriction site map of the Bacillus licheniformis chromosomal region bearing the cloned fragment is constructed.

[Isolation and characteristics of a plasmid from the thermotolerant strain of Bacillus licheniformis]. / Vydelenie i kharakteristika plazmidy iz termotolerantnogo shtamma Bacillus licheniformis.

A 8.3 kb cryptic plasmid was isolated from the thermotolerant strain of Bacillus licheniformis 28KA and designated pLT83. The replicative (rep) region was localized on the plasmid map. The pLT83 plasmid labelled in vitro with an antibiotic resistance determinant is able to replicate in B. subtilis cells. The pLT83 plasmid replicates stably in B. licheniformis strain at higher temperatures (37-60 degrees C) than in B. subtilis cells (37-50 degrees C). The plasmid and its derivatives may be used as vectors for gene cloning in B. subtilis and B. licheniformis cells.

[Cloning of genetic material in Bacillus]. / Klonirovanie geneticheskogo materiala v batsillakh.

Plasmid vectors capable for propagation of Bacillus subtilis DNA fragments containing riboflavin genes were constructed. Cloning of rib operon using pUB110 derivatives was performed in recE4 strain by using sequentional rescue of plasmids containing subfragments of the operon. Also, rib operon was cloned on the vectors containing DNA repeats. It was shown that the presence of direct and inverted repeats within plasmids allows to transform B. subtilis cells by monomers of plasmid DNA. Vectors that contained repeated sequences of DNA and ensured efficient cloning of genetic material in B. subtilis recipient cells were constructed. The use of streptococcal plasmid pSM19035 allowed to obtain vectors which were suitable for cloning large DNA fragments (6 MD and even more) in B. subtilis. A model of B. subtilis transformation by various types of plasmid DNA is presented. The model is in agreement with the general conception of chromosomal DNA transformation.

[Cloning the operon genes of riboflavin biosynthesis in Bacillus subtilis on plasmid vector pBR322 in Escherichia coli]. / Klonirovanie genov operona biosinteza riboflavina Bacillus subtilis na plazmidnyi vektore pBR322 v Escherichia coli.

The operon for riboflavine biosynthesis of Bacillus subtilis wild type and its operator-constitutive derivative have been cloned in Escherichia coli cells on the plasmid pBR322 vector. The plasmids constructed were able to transform strains of E. coli and Bac. subtilis from Rib- to Rib+ phenotype. A DNA insert into the EcoRI site of pBR322 causes a decrease in tetracycline gene expression. The operator of the riboflavine operon of Bac. subtilis does not participate in regulation of the operon expression in E. coli cells.

[Artificial Tn2551 transposon with replicative properties]. / Iskusstvennyi transpozon Tn2551 s replikativnymi svoistvami.

A hybrid plasmid, pBE10 was constructed. It consists of DNAs of RSF2124 (ColE1 :: Tn3) plasmid and pUB110 plasmid of Staphylococcus aureus. The latter can be stably maintained in Bacillus subtilis. BamHI cleaved pUB110 was introduced into the BamHI site of transposon Tn3 and the resulting enlarged Tn3 (Tn2551) was transposed from pBE10 onto phage lambda and than to pMB9 (Tc) and RSF1010(Sm Su) plasmids. Restriction and heteroduplex analysis of pMB9 :: Tn2551(pBE21) and RSF1010 :: :: TN2551(pBE32) was carried out. Plasmids pBE10, pBE21 and pBE32 demonstrated some kind of molecular instability when introduced by transformation into Bacillus subtilis.

[Isolation and properties of merodiploid strains with F-factor including the pts-region of the Escherichia coli K-12 chromosome]. / Vydelenie i svoistva marodiploidnykh shtammov s F'-faktorom, vkliuchaiushchim pts-oblast' khromosomy Escherichia coli K-12.

A technique of hybridization of haploid methanol-utilizing yeast Pichia pinus MH4 is worked out using UV- and N-nitrosoguanidine-induced auxotrophic mutants. Vegetative diploid cultures are isolated. Tetrad analysis and random spore analysis have revealed a meiotic nature of spores, recombination of genetic material in the process of sporulation and the chromosomal nature of some mutations. A possibility to construct a genetic map of the yeast Pichia pinus MH4 is demonstrated on the basis of tetrad analysis. Three linkage groups are revealed. The life cycle in a homothalic haploid yeast, Pichia pinus, was demonstrated. They are capable to form zygotes and meiotic spores under conditions preventing vegetative growth.