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Biomedical subjects

A I Stepanov

Publications and source records attributed to A I Stepanov.

At least 37 records · Page 2Linked to original sources
PubMed1982

[Location of the traS- and finO-type genes and of the oriT region of plasmid R6K].

The regions of B6K DNA corresponding to oriT, finO and traS are mapped using a number of hybrid plasmids and deletion mutants pAS3::Tn5, pAS3::Tn7 and pAS3::Tn9 obtained in vitro after treatment with restriction endonucleases EcoRI and BamHI. FinO- and traS-like genes were mapped in the regions of 10 and 25,0-25,4 MD, respectively, oriT being situated in the region of 4,6-4,7 Md.

Chromosome Deletion↗
PubMed1982

[Bacillus subtilis mutants resistant to roseoflavin].

Bacillus subtilis mutants resistant to 100 mkg/ml of roseoflavin/8-dimethylamino (nor) riboflavin/have been shown to excrete 0.5 to 20 mkg/ml riboflavin and small amounts of FMN and FAD into the culture medium. The rosR mutations are localized in the operator region of riboflavin operon. The combination of rosR and ribC mutations (the latter being mutation in the regulator gene) leads to hyperproduction of riboflavin.

Anti-Bacterial Agents↗
PubMed1982

[Bacillus licheniformis as an object for the propagaton of heterologous genetic material from bacilli].

Bacillus licheniformis was transformed with plasmids pUB110 and pJJ10 (pUB110 - pBR322) isolated from Bac. subtilis and Escherichia coli, respectively. It was revealed that the structure and genetic properties of the plasmids did not change during the transformation process. pJJ101 (pJJ10-rib) DNA isolated from E. coli and containing helper pJJ10 plasmid was used, as a recipient. It was shown that pJJ101 rib markers were "rescued" by the resident plasmid during transformation of Bac. licheniformis (pJJ10). Plasmid pLP1 containing ribB, ribD, Kmr genes and the pUB110 replicator, was isolated from the transformants. pLP1 plasmid might be considered as a detected derivative of the parental pJJ101 plasmid. The deletion is presented by 3,9 MD segment that contains the pBR322 replicator. pLP1 DNA is capable of transforming plasmidless strains of Bac. licheniformis and Bac. subtilis.

Bacillus↗
PubMed1981

[Genetic control of plasmid R6K conjugativity].

The regions determining conjugation ability of plasmid R6K were localized by means of deletion mutants obtained in vitro and in vivo and Tra- mutants induced by integration of transposons Tn5, Tn7 and Tn9 into different DNA sites of the conjugative deletion mutant pAS3. At least 13 genes were found to be involved in the genetic control of R6K conjugativity, on the basis of genetic, restriction and heteroduplex studies. They were mapped within the two DNA regions having the total molecular weight of about 10-11 md. A transposon-like structure with a replication function has been located between them.

Chromosome Mapping↗
PubMed1981

[Artificial Tn2551 transposon with replicative properties].

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.

Bacillus subtilis↗
PubMed1979

[Riboflavin auxotrophs of Escherichia coli].

Escherichia coli riboflavin auxotrophs having a different level of riboflavin requirement were isolated. This auxotrophic mutations are located near cysB93 and trpA62 markers. The complementary effect of Bacillus subtilis riboflavin operon linked with pPR1 hybrid plasmid with rib8-1 and rib1-1 mutations was obtained.

Bacillus subtilis↗
PubMed1979

[Hybrid plasmid pSD1 containing the immunity region of bacteriophage lambda].

Hybrid plasmid pSD1 carrying the immunity region of the coliphage lambda and bio operon have been obtained by means of studying the efficiency of transcription DNA fragments in the plasmid RSF2124. The molecular weight of this plasmid is 17.2 Md. The growth inhibition of phage lambdavir has been observed in cells carrying the new hybrid plasma. The properties of the plasmid pSD1 and probable reasons of the growth inhibition of phage lambdavir are discussed. The hybrid plasmid pSD2 carrying genes R, A and J of phage lambda has been constructed on the basis of the plasmid RSF2124. There are cohesive ends in this plasmid which make possible its packing in the phage lambda head. Hybrid plasmid pSD3 carrying genes P and Q of phage lambda has also been constructed.

Bacteriophage lambda↗
PubMed1979

[Nature of the compatible inheritance of hybrid plasmid pAS8 and its deletion mutants with replicon ColE1].

Hybrid plasmid pAS8, that consists of RP4 and ColE1 replicons, is incompatible with RP4 but co-exists with ColE1 replicon. The deletion mutants of pAS8, that replicates under the control of ColE1 replicon only, are incompatible with ColE1 derivatives, although the copy number of pAS8 and its deletion mutants in the cell is the same (1-3 per the chromosome). Incompatibility effect of plasmids is expressed in a sharp decrease in transformant's yield during selection of incoming and resident plasmids markers. In this case incompatibility is a very fast process, that leads to the elimination of resident or incoming plasmid just before plating on the selective medium. On the base of negative control theory, the repressors yield, synthesized in the presence of 1-3 copies of the deletion mutant is enough for the expression of ColE1-specific incompatibility. This ColE1 incompatibility is probably connected with the functional activity of ColE1 replicon. When ColE1 factor replicates under the control of RP4 replicon the expression of ColE1-specific incompatibility does not occur. Possibly, the incompatibility effect takes place when pAS8 deletion mutants cause the synthesis of ColE1-specific repressor. Also, the replicons of ColE1 may competein the membrane attachment site.

Bacteriocin Plasmids↗
PubMed1978

Mapping of RP4 plasmid using deletion mutants of pAS8 hybrid (RP4--ColE1).

We used the hybrid plasmid pAS8 in order to conduct the genetic analysis of RP4 plasmid. The presence of two replicons in the hybrid plasmid permitted to expand the spectrum of deletion mutants of RP4 isolated, which are capable to autonomous replication. The shortening of the hybrid plasmid was achieved by P22 transduction, by induction of deletion mutants using mitomycin C, as well as by seletion of Tra- mutants on the basis of resistance of cells to P-specific phages. These techniques have lead to isolation of clones possessing different combinations of plasmid resistance determinants. Comparison of phenotypic characteristics of deletion plasmids pAS9, pAS10, pAS11, pAS12 and pAS10-2 permitted to propose the map for pAS8 plasmid with the following sequence of markers: tra--kam--ColE1--amp--tet... Heteroduplex analysis of deletion mutants of pAS8 permitted to construct a physical map and to elaborate in greater detail the functional map of RP4 plasmid. The correlation between the ability of mutants to replicate in polA(TS) strain at nonpermissive conditions and the length of the deleted segment permitted to map rep genes of RP4 on a region with coordinates 9.8--17.3 kb. A relationship between the manifestation of incompatibility of mutants with Inc P-1 plasmids and the length of deletions points out that inc genes are located on DNA region with coordinates 2.1--9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic independence of the replication control and incompatibility control in RP4 plasmid.

DNA, Bacterial↗
PubMed1978

[Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells].

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.

Bacillus subtilis↗
PubMed1978

[Genetic and heteroduplex analysis of deletion mutants of plasmid pAS8].

Heteroduplex analysis of deletion mutants of plasmid pAS8 permitted to construct a physical map and to elaborate in greater detail the genetic map of RP4 plasmid. The correlation between the ability of mutants to replicate in cells lacking functional DNA-polymerase I and the length of the deleted segment permitted to map rep genes of RP4 on DNA region with coordinates 9.8-17.3 kb. A relationship between the manifestation of incompatibility of mutants with IncP-1 plasmids and the length of deletions indicates that inc genes(s) are located on DNA region with coordinates 2.1-9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic non-identity of the replication control and incompatibility control in RP4 plasmid. Different degrees of incompatibility manifested by different plasmids suggest a possible polygenic control of the incompatibility.

Chromosome Deletion↗