[Preparation of functioning recombinant (hybrid) DNA molecules in vitro (genetic engineering experiments)].

In this paper we describe the preparation of hybrid plasmid consisting of ColE1 DNA and DNA of R6K plasmid. ColE1 plasmid represents a circular DNA with the molecular weight of 4,2-10(6) daltons. It determines colicine synthesis E1, has relaxed control of replication and is present in the cell in several dozens copies. Transmissive plasmid B6K represents a circular DNA molecule with the molecular weight of 28-10(6) daltons. It confers the resistance to amplicillin and streptomycin and belongs to the compatibility group X. This plasmid also has relaxed control of replication (up to 30 copies per cell). Circular superhelical DNA of ColE1 was prepared after it is amplification with cloramphenicol according to Clewell by ultracentrifugation in CsCl-EtBr density gradient. The yield of superhelical ColE1 DNA in our experiments was up to 300 mg/gram cells. Circular superhelical R6K DNA was isolated from E. coli strain J53 R6K grown to stationary phase in the presence of 15 mg/ml of streptomycin. The yield of superhelical DNA was equal to about 45 mg per 1 gram cells. EcoR1 restrictase was prepared from E. coli KM182 as described by Thomas et al. From 37 grams of cells 10 ml of enzyme solution was prepared. 1 ml of this solution was able to restrict completely 2 mg of ColE1 DNA during Hu incubation for 30 degrees min at 37 degrees C. Restrictase activity was analyzed by electrophoresis of ColE1 DNA restricts in 0,7% agarose gel as described by Tanaka. DNA ligase was prepared from E. coli B. cells infected by T4 phage am81 mutant as described by Weiss. The activity of enzyme solution was equal to 90 units/ml (according to Richardson). In standard "hybridization" experiment 4 mug of ColE1 DNA and 4 mug of R6K DNA were incubated for 1 hour at 37 degrees in 0,2 ml of the solution containing 40 mM tris-HCl, pH 7,4; 10 mM MgCl2 50 mM NaCl, 10 mM mercaptoethanol and 5 mul of EcoR1 restrictase. The reaction was terminated by heating the reaction mixture to 65 degrees C for 5 min. In accordance with the results of others CoLE1 DNA gave one restrict and R6K DNA-two restricted fragments. 100 mul of the restricted mixture was chilled to 0 degrees and the following ingredients were added 10 mul of 50 mM MgCl2; 10 mul of 100 mM dithiotreitol; 10 mul of 0,5 mM ATP, 20 mul of water and 8 mul of ligase solution (90 units/mul). The mixture was incubated at 0-0,5 degrees for 53 hours, thereafter it was used for the transformation of E. coli C600 according to Tanaka et al. Several classes of hybrid molecules due to different combinations of restricted fragments of parental species were expected. These molecules had to confer the immunity to colicine E1 and to carry the defect in the gene controlling colicine synthesis since EcoR1 restrictase splits ColE1 DNA in the region of corresponding gene. In addition, certain classes of transformants with hybrid DNA molecules had to be resistant to one or both antibiotics as determined by the R6K plasmid portion...