[Controlled fragmentation of DNA. I. Acentric hydrodynamic breakage and a possibility for the isolation of T5 gene groups].
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Biomedical subjects
Publications and source records attributed to A S Solonin.
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With the use of the strain-overproducer restriction endonuclease R.EcoRV was isolated and purified to homogeneity. The molecular mass of the enzyme was determined by gel filtration and polyacrylamide gel electrophoresis to be 25 000 daltons. According to the data of immunological tests R.EcoRV differs in its antigenic characteristics from restriction endonucleases R.EcoRI and R.EcoRII. Dependence of enzyme activity on pH, ionic strength, temperature, presence of divalent cations (Mn2+, Mg2+, Co2+, Zn2+, Ni2+ and Cd2+) and organic solvents (glycerol, dimethylsulfoxide, ethanol) has been studied. It was shown that under conditions of replacement of Mg2+ for Mn2+ or after addition of organic solvents relaxation of R.EcoRV specificity takes place. It was shown also that R.EcoRV is able to digest T-even bacteriophage DNAs with different types and extents of modification. DNA modified by the action of MR.EcoRV system in vivo is susceptible to R.EcoRV in vitro. Under conditions of relaxed specificity noncanonical sites are susceptible to R.EcoRV attack. The fragments resulted may be cloned in canonical pBR322 EcoRV site.
A collection of recombinant plasmids directing the increased synthesis of the cro-repressor of lambda imm434 bacteriophage under control of the lacZ promoter of Escherichia coli has been constructed. About 87 000 molecules of cro-repressor monomer per cell, e. g. 0,7% of total cell protein are synthesized by the overproducers. Strain bearing one of such plasmids--pBS71-1. Several recombinant plasmids in which the cro-gene is placed under the control of early genes promoter of lambda phage (pl, pr, prm) have been also prepared. Plasmid pIL206, constructed on the basis of pIL203 vector, was used as a source of lambda-promoters.
Various clinical strains of Enterobacter cloacae have been examined for the presence of site-specific endonuclease activities. Restriction endonucleases of class II have been isolated from six strains. Recognition sequences for all of these endonucleases were determined and the cleavage sites were identified for two of them. The enzymes prove to be isoschizomers of EcoRII and PstI. Restriction endonucleases Ecl2zI and Ecl37kI recognise the nucleotide sequences CTGA/G and are true isoschizomers of PstI. The genes for Ecl54kI and Ecl57kI restriction-modification systems (isoschizomers of EcoRII) were found to be located on the IncN group plasmids, whereas the genes for Ecl2zI and Ecl99kI seem to be located on the chromosomes of host cells.
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