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Biomedical subjects

A N Kravets

Publications and source records attributed to A N Kravets.

16 recordsLinked to original sources
PubMed1998

[Novel isoschizomers of restriction endonucleases from Pseudomonas aeruginosa strains].

Testing of Pseudomonas aeruginosa strains from the collection of the L. V. Gromashevsky Institute of Epidemiology and Infectious Diseases (Kiev) for specific endonucleases resulted in isolation of five class II restriction endonucleases, which were partially purified and their recognition targets were determined. Two of these endonucleases, Pae2kI and Pae18kI, are isoschizomers of Bg1II (5'-AGACTC-3'). Pae5kI and Pae14kI recognize the 5'-CCGC/GG-3' sequence and are therefore true isoschizomers of SacII. Hence, Pae17kI is an isoschizomer of PvuII and cleaves the DNA within the recognition sequence 5'-CAG/CTG-3'. Bg1II and PvuII are for the first time detected in Pseudomonas aeruginosa.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1998

[High homology of plasmids carrying class II restriction modification systems, EcoRV isoschizomers, and their prevalence in natural Escherichia coli strains].

Screening of 660 clinical Enterbacteriaceae strains from the collection of L. V. Gromashevsky Kiev Institute of Epidemiology and Infectious Diseases for specific endonuclear activity revealed site-specific endonucleases, EcoRV isoschizomers in 6 E. coli strains. Genes coding for endonucleases and methyltransferases were localized on small (6.2 kb) multicopy Hsd+ plasmids. All plasmids were successfully transferred in laboratory strain E. coli K802. Restriction analysis and subcloning showed no differences in the structural and functional organization of studied plasmids and a previously revealed pLB1 plasmid [3,10], this reflecting their high homology, if not identity. These data allow us to propose effective horizontal transfer of EcoRV plasmids among natural E. coli isolates in the region studied.

Chromosome Mapping↗
PubMed1983

[Possible dependence of the results of phage typing Ps. aeruginosa strains on the presence of R plasmids].

The plasmid composition of 209 strains of Ps. aeruginosa was determined. The strains were isolated from patients, animals and environment in different geographical areas. The number of the plasmid-containing strains averaged 26.8 per cent. The molecular weights of the plasmids varied from less than 10 to more than 150 MD. 41 conjugative plasmids were transmitted to the recipients of Ps. aeruginosa RAO 303. 66 per cent of them had a restrictive effect on the development of phages used in genetic studies, epidemiological phage typing (Lindberg Collection), and medical practice. This resulted in the changing of the phage type of the host strain. Similar results were obtained in the studies with 10 standard R plasmids representing different incompatibility groups. No relation between the spectrum of the phage restriction, group specificity and the other properties of the plasmids was observed. About 50 per cent of the plasmids markedly lowered the sensitivity level of Ps. aeruginosa RAO 303 to the therapeutic pyocyanic phage. The systems of restriction and modification of DNA coded with plasmids were not detected. A possible changing of the phage type of Ps. aeruginosa strains under the effect of R plasmids should be considered in epidemiological assays and respective treatment measures.

Bacteriophage Typing↗
PubMedDate not supplied

[Isolation of highly active strain producing the antistaphylococcal antibiotic batumin].

The use of chemical and UV-induced mutageneses allowed us to increase the biosynthetic activity of the strain capable of producing new antistaphylococcal antibiotic, batumin. The strain of Pseudomonas batumici N17 producing 87-100 mg batumin per liter culture liquid was selected. Its activity was 3.5-5 times higher than the activity of the most potent natural strain. P. batumici N17 was shown to be stable in relation to the synthesis of batumin.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Transposon mutagenesis in preparing mutants of a strain--a producer of a new antibiotic batumin].

Various plasmids carrying transposon Tn5 were used to generate insertion mutants synthesizing batumin, a unique antibiotic with a selective antistaphylococcal effect. One of the plasmids used provided a sufficient yield of the clones in question. An analysis of over 7000 clones allowed us to select the mutant clones with increased and decreased levels of batumin synthesis and the mutants that lost the ability to synthesize this antibiotic.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Plasmid localization and cloning of restriction modification genes from Citrobacter freundii 4111 strain].

Over 60 producing strains of restriction endonucleases type II have been found among 500 different strains, mostly Enterobacteriaceae. The strain Citrobacter freundii 4111 produces restriction endonuclease CfrBI, a new isoschisomer of StyI. The genes of the restriction-modification system CfrBI were located on the multicopy plasmid pZE8 containing the Co1E1-type replicon and cloned to E. coli K802. The deletion variant of 3.2-kb pZE8 which contains intact restriction-modification and a DNA fragment responsible for autonomous plasmid replication was selected among the recombinant plasmids. The strain with higher R. CfrBI production (at least 10,000,000 U/g cells, which is 500-fold higher than the wild strain) was constructed.

Citrobacter freundii↗
PubMedDate not supplied

[Properties of the replicator region of the natural plasmid pLG13 containing genes of the EcoRV restriction-modification system].

The previously constructed plasmid pILRV8 that induces endonuclease EcoRV gene overexpression kills cells of some E. coli strains under the induction of this enzyme synthesis. Cell transformation by natural plasmid pLG13 carrying genes of the EcoRV restriction--modification system was found to appreciably enhance cell viability ("survival") under endonuclease overproduction. A plasmid pLG13 region located in immediate proximity to the methylase gene was shown to be responsible for the above effect. This region was also capable for autonomous replication. The analysis of the DNA primary structure in the found replicator region allowed to refer the pLG13 to ColE1 family plasmids. Perturbations in the region lead to loss of the "survival" effect and change of the plasmid replicative properties. A relationship between the replicon elements, the EcoRV genes region and "survival" effect is discussed. Based on the replicon found multicopy vector molecules have been constructed.

Base Sequence↗
PubMedDate not supplied

[Cloning and regulation of gene expression of EcoRV restriction- modification system].

A number of recombinant plasmids, containing EcoRV restriction-modification genes have been constructed. Individual genes of this system were introduced into plasmids of various incompatibility groups. Promoter regions of genes encoding methylase and restrictase have been cloned and studied. With the use of specialized vector pVE8 it was shown that the efficiency of the endonuclease gene promoter is comparable with early lambda phage promoters and produced about 70% of PL efficiency. The efficiency of the methylase gene promoter region was twice less than the efficiency of the restriction endonuclease gene promoter. Plasmid with restriction endonuclease gene promoter located downstream in relation to the additional regulatable phage lambda promoter PL has been obtained. It enabled us to construct strains 30-40 fold overproducing this enzyme under conditions of inactivation of the temperature sensitive phage repressor c1857. This construction directs the production of a high level (10%) of the total cellular soluble proteins) of the EcoRV restriction enzyme. The factors that influenced the level of enzyme synthesis under induction are discussed.

Base Sequence↗
PubMedDate not supplied

[A system of EcoRV restriction-modification: genes, enzymes and synthetic substrates].

The genes, encoding the restriction endonuclease and modification methylase EcoRV have been cloned from the natural plasmid pLG13 into pBR32 derivative vector pIL233. A resultant clone, expressing both enzyme activities, was used as a source of DNA for sequencing these genes by a procedure, that employed construction of deletion derivatives used to locate borders (by means of a functional test) and to sequence ca. 300 bp near the deletion breakpoint. From the sequence data, we infer that the endonuclease, a 29 KDa protein, and the methylase, a 36 KDa protein, are transcribed from a 310 bp intergenic region in opposite directions. There is no apparent homology between the enzymes and genes of the EcoRI and the EcoRV systems. A synthetic decamer, containing the EcoRV endonuclease recognition sequence and a phosphoamide bond at the cleavage point, is not cleaved by the highly purified endonuclease; the unmodified synthetic decamer is cleaved at the same conditions, only that the cleavage occurs to produce a blunt end--GAT/ATC, and not in a place previously reported (GATAT/C).

Base Sequence↗
PubMedDate not supplied

[EcoRV restrictase: physical and catalytic properties of homogenous enzyme].

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.

Base Sequence↗
PubMedDate not supplied

[Biosynthesis of the new antistaphylococcal antibiotic batumin].

Levels of batumin biosynthesis by separate cells of the antibiotic-producing strain were investigated. The strain belongs to the genus Pseudomonas. It was found that the cell population was not homogeneous by that property. There were isolated highly active, active and low active clones. Specific selection of the most active clone made it possible to increase the number of the cells with high levels of the antibiotic biosynthesis in the population from 8.8 to 84.7 per cent. A clone was isolated which formed no zones of the growth inhibition of Staphylococcus aureus 209P. The data showed that the biosynthesis of batumin was genetically unstable which could be indicative of the plasmid localization of the genes controlling its production. However, with the basic methods of the plasmid DNA isolation it was demonstrated that the batumin-producing strain contained no extrachromosomal DNA which indicated to the chromosomal localization of the genetic locus responsible for the antibiotic synthesis.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Restriction system--type II modification in Enterobacter cloacae strains].

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.

Chromatography, DEAE-Cellulose↗