[Determinant of resistance to kanamycin in Streptomycin rimosus: amplification in the chromosome and reversible genetic instability]. / Determinant ustoichivosti k kanamitsinu Streptomyces rimosus: amplifikatsiia v sostave khromosomy i obratimaia geneticheskaia nestabil'nost'.

The study on the kanamycin resistance determinant (Kanr) in an oxytetracycline--producing strain of S. rimosus showed that it was capable of amplifying in the chromosome during selection for increasing the antibiotic resistance level. The amplification of the DNA fragment with a molecular weight of 10.3 MDa containing Kanr amounted to 300 copies per genome, which resulted in a more than 1000-fold increase in kanamycin resistance level. Cloning of the Kanr determinant on plasmid SLP1.2 in S. lividans strain 66 was performed. In Streptomyces lividans strain 66 the Kanr determinant preserved the capacity for amplification in the hybrid plasmid pSU10 integrated into the chromosome. The Kanr determinant in the strains of S. rimosus and S. lividans was characterized by transfers Kanr in equilibrium Kans with a frequency of 1 X 10(-3). It was shown that the mutation in S. lividans strain 66 resulting in phenotype Kans was not connected with the structural Kanr gene on plasmid pSU10 but was localized on the chromosome. Phenotype Kans was promoted by a decrease in the number of the copies of the regulatory genetic element designated RES1. The reverse to phenotype Kanr might be due to one of the following events: amplification to the initial level of RES1 and amplification up to 200 copies per the genome of the hybrid plasmid pSU10 containing the Kanr determinant. Amplification of the Kanr determinant with preserved initial level of RES1 element resulted in a more than 1000 times increase in the resistance level.

[DNA cloning in actinomycetes: the creation of vector systems]. / Klonirovanie DNK u aktinomitsetov: sozdanie vektornykh sistem.

A new vector type was constructed on the basis of SLP 1.2 plasmid and the Kanr determinant of S. rimosus P3. It was shown that the determinant was capable of amplifying in the chromosomes of S. rimosus during its improvement for increasing the level of resistance to kanamycin. Cloning of the Kanr determinant was performed on the Pst I-A fragment of the SLP 1.2 plasmid in S. lividans 66. The Kanr determinant was expressed in the resulting hybrid plasmids, e. g. pSU 3, thus providing resistance of S. lividans to 20 micrograms/ml of kanamycin. As a result of repeated passages variants capable of growing in the presence of 50 000 micrograms/ml of the antibiotic were selected. The electrophoretic analysis of the total DNA fragments obtained after exposure to different endonucleases showed that they were identical to the respective fragments of the hybrid pSU 3 plasmid and amounted to approximately 40 per cent of the total DNA. The presence of the unique sites for the Bam HI, ClaI and SacI restriction endonucleases on the pSU 3 plasmid in an insignificant area and the relative stability (30-100 per cent) of the amplified variants allowed using it for cloning and amplification of DNA in Streptomyces. Plasmids were identified with the genetic and physical methods in 13 strains of the blue systematic group among the 72 strains studied. A multicopy plasmid with a molecular weight of 5.6 MD designated as pSB 24.1 was identified in the family of the plasmids of S. cyanogenus. The deletion and insertion variants of the pSB 24.1 plasmid were obtained. The comparative study of the properties of these plasmids revealed the areas insignificant for replication and maintenance of the pSB 24.1 plasmid and the area determining the Ltz+-phenotype. It was suggested that formation of the deletion and insertion variants of the pSB 24.1 plasmid was provided by the site specific recombination mechanisms. The presence of the unique sites for a number of the restriction endonucleases located in the insignificant area, the presence of the selective marker and the high transformation frequency allowed one to consider the multicopy pSB 24.1 plasmid an acceptable vector for cloning of DNA in Streptomyces.