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A I Stepanov

Publications and source records attributed to A I Stepanov.

At least 55 records · Page 3Linked to original sources
PubMed1978

[Genetic heteroduplex analysis of the R6K plasmid].

The results of genetic studies of R6K Tra1- and R6Kdelta[Sm1] mutants of R6K plasmid and those of heteroduplex analysis of DNAs have shown that DNA of this drug-resistant factor contains three loops flanked by the inverted repeats. The latter are designated as IR1, IR2 and IR3 and are of 50, 100 and 120 nucleotides in size respectively. IR1 is inserted into the loop flanked by IR2. Loops with these two repeats are located in major EcoR1 fragment, IR3 having been found in minor EcoRI fragment of the plasmid. The evidence obtained from the analysis of heteroduplex R6K/RSF2124 has shown that the loop with IR1 is corresponding to transposon Tn3. The extent of the deletion deltaSm1 indicates that IR2 may be a part of a transposon bearing the resistance to streptomycin. By comparing present data with those obtaine from the analysis of the RSF1040 factor of DNA replication initiation sites (Grosa et al., 1976), it has been suggested that the loop with IR3 represents a transposon with replicative functions (TnRep). The deletion of the mutant plasmid R6Kdelta[Sm1] (7.2 . 10(6) daltons in size) which affected one of the EcoRI sites not only confers the sensitivity to streptomycin but enhances also the efficiency of conjugational transfer and results in the loss of the R6K ability to bring about integrative suppression and to inhibit the fertility of the plasmids from IncP and IncN groups. The deletion mutant proved to have lost the property of incompatibility with the initial plasmid R6K and with itself.

Conjugation, Genetic↗
PubMed1977

[Analgos of riboflavin, lumiflavin and alloxazine derivatives. II. Effect of roseoflavin on 6,7-dimethyl-8-ribityllumazine and riboflavin synthetase synthesis and growth of Bacillus subtilis].

The replacement of 8-CH3 group in the riboflavin molecule results in the formation of specific antimetabolites. They are rozeoflavin, 7-desmethylrozeoflavin, 8-amino (nor) riboflavin, 8-ribitylamino (nor) riboflavin. Effect of rozeoflavin and other riboflavin analogues on the growth and regulatory characteristics of Bacillus subtilis strains with different genetic state of riboflavin operon is studied. Roseoflavin at a concentration of 0.05 mkg/ml inhibits DRL synthesis in rib-b110 strain. An analogue inhibits the growth of auxotrophic and prototrophic strains at concentrations of 0.5 mkg/ml and 50 mkg/ml respectively. Riboflavin (1 mkg/ml) recovers the growth of bacteria. The curve of rozeoflavin regulation of DRL and riboflavin synthetase synthesis is shifted in 100 times in the direction of lesser concentrations as compared with riboflavin and 8 amino (nor) riboflavin. 180 mutants resistant to 100 mkg/ml of rozeoflavin were selected. 150 mutants over-synthetize riboflavin.

Bacillus subtilis↗
PubMed1976

[Transmissible hybrid plasmid RP4-ColE1].

Hybrid plasmid RP4-ColE1 was obtained by joining DNA plasmids RP4 and ColE1, each of which possessed only one site of restriction for EcoR1. These plasmid molecules were restricted by endonuclease EcoR1 and then treated by ligase. The hybrid plasmid retained the property of transmissibility typical for drug factor resistance RP4. Non-transmissible mutant of the hybrid plasmid selected by the character of the resistance of Escherichia coli C600 (RP4-ColE1) to the phage PRR1 is used for subsequent investigations.

Colicins↗
PubMed1975

[Eremothecium ashbyii mutants resistant to 2,6-diaminopurine].

3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.

2-Aminopurine↗
PubMed1975

[Eremothecium ashbyii mutants resistant to 8-azaguanine. II. Mutants with different degrees of resistance to 8-azaguanine].

Guanine, unlike adenine and hypoxanthine, can not eliminate the inhibitory effect of adenine analogues on the growth and flavinogenesis of Eremothecium ashbyii. Guanine does not restore riboflavin synthesis inhibited with 5-10(-3) M 8-azaguanine. Low adenine concentrations (10(-4)-3-10(-4) M), which do not influence the inhibitory effect of 5.-10(-3) M 8-azaguanine, restore the riboflavin synthesis in combination with guanine. On the basis of the data obtained as well as the data of biochemical analysis it is concluded that the riboflavin producer studied lacks guanosinemonophosphate reductase. The mutants resistant to various concentrations of 8-azaguanine have been obtained. In all mutants resistant to 8-azaguanine the efficiency of the incorporation of 14C-guanine and 14C-adenine into mycelium is decreased as compared with the susceptible strain. The mutant Azg-R 10 resistant to high (3-10(-3) M) concentrations of 8-azaguanine, 8-azaadenine and 2,6-diaminopurine secretes inosine-like compounds when grown in a synthetic medium. The stepwise increase of the mutant resistance to 8-azaguanine from 10(-4) M TO 3-10(-3) M did not result in further enhancement of riboflavin synthesis.

Ascomycota↗
PubMed1975

[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...

Colicins↗
PubMed1975

[Riboflavin and lumiflavin analogs and alloxazine derivatives. I. Effect on riboflavin synthesis by and growth of Bacillus subtilis].

Effect of riboflavin, lumiflavin and a number of alloxazine derivatives is studied on the growth of three Bacillus subtilis strains with different genetic state of riboflavin operon as well as their ability to show coenzyme and regulating properties characteristic of vitamin B2. The fission of the ribityl chain of the riboflavin molecule results in the complete loss of both coenzyme and regulating properties of vitamin B2 (analogues of lumiflavin and alloxazine derivatives). Different types of esterification of hydroxyl groups of ribityl chain or its replacement by dulcityl chain results in the decrease of regulating properties characteristic of riboflavin as an end product of biosynthetic pathway (tetraacetylriboflavin and its derivatives, tetrapropionyl- and tetrabutyrylriboflavin) or to the loss of coenzyme and regulating activities (galactoflavin and tetrabenzoylriboflavin). The length increase of hydrocarbon chain under esterification of hydroxyl groups in ribityl chain results in enhancing vitamin B2 non-specific inhibitory effect on the growth of Bacillus subtilis strains studied. The replacement of 8-CH3 group in the riboflavin molecule by amino- or D-ribitylaminogroup results in the formation of specific antimetabolites, 8-amino(nor)riboflavin and 8-D-ribitylamino(nor)riboflavin respectively. Depending on the nature of other alterations or replacements of the 8-CH3 group of riboflavin molecule and their combinations with alterations of ribityl chain the reduction of the regulating activity (8-alpha-bromtetraacetylriboflavin) or a sharp decrease of coenzyme and regulating functions (analogues N 9 to N 15) takes place. The replacement of riboflavin-5-N-oxide and 2-thioriboflavin type did not affect the studied properties of riboflavin.

Bacillus subtilis↗