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A A Prozorov

Publications and source records attributed to A A Prozorov.

At least 37 records · Page 2Linked to original sources
PubMed1992

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology.

To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between ts pE194 derivatives carrying segments of the chromosomal beta-gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for beta-gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 bp of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.

Base Sequence↗
PubMed1992

[Determination of the minimal length DNA homologous region required for plasmid integration into the Bacillus subtilis chromosome via homologous recombination].

With a view to determine a minimal sequence length of homology necessary for RecE-dependent homologous recombination in Bacillus subtilis cells, we developed a system, based on interaction between plasmid replicon and bacterial chromosome. Recombination frequencies were measured between ts plasmid pE194 derivatives carrying chromosomal beta-glucuronidase gene (bglS) fragments of various length, and a bacterial chromosome. The homologous recombination events resulted in bglS gene disruption. Approx. 70 bp of homology were found to be necessary for detectable homologous recombination. Homologous recombination was not detected when homology was equal 25 bp. These data indicate that homology requirement for recombination in B. subtilis differs from that in Escherichia coli.

Bacillus subtilis↗
PubMed1992

[Bacillus subtilis gene rec223: molecular cloning and proposed function of its protein product].

Chromosomal DNA fragment which complemented rec223 mutation of Bacillus subtilis was cloned. Introduction of one copy of the cloned gene into the cells of the rec mutant restored both normal activity for DNA damages repair after mitomycin C action and recombination proficiency. Using multicopy vector led to no formation of recombinants, which was probably connected with overproduction of rec223 gene protein product in Bacillus subtilis cells.

Bacillus subtilis↗
PubMed1991

[Isolation of Rec mutants of Bacillus subtilis via insertional mutagenesis].

A number of mutants supersensitive to mytomycine C were isolated via insertion of Tn917 into Bacillus subtilis chromosome. Six Rec mutants with lower transformation frequency, though with nondisturbed capacity of DNA binding and uptake, were isolated. Two of these mutations are situated in those chromosomal sites where location of Rec genes had not yet been registered.

Bacillus subtilis↗
PubMed1991

[Transfer of transformation and conjugation plasmids from Escherichia coli to Bacillus subtilis and Azospirillum brasilense].

The conjugative transfer of RP4 plasmid from Escherichia coli to Azospirillum brasilense was detected after introduction and subsequent incubation of these microorganisms in soil. The plasmid transfer via transformation from Escherichia coli to Bacillus subtilis was observed in case both bacteria were growing together in sand containing sucrose solution. The possible reason for low frequency interspecies plasmid transformation under conditions close to natural habitats is poor survival of "domesticated" rather than wild type Bacillus subtilis strains and lack of competence state in this case.

Azospirillum↗
PubMed1991

[Features of transformation of competent cells and Bacillus subtilis protoplasts by integrative vectors].

The effect of structural peculiarities of DNAs from integrative plasmids on the transformation activity was studied. Monomeric forms of the plasmids can only transform B. subtilis competent cells, when plasmid selective marker is inserted into chromosomal fragment within the plasmid. Polymeric forms are needed for efficient transformation. Both single- and double-stranded DNAs of integrative plasmids transform no B.subtilis protoplasts, this being irrespective of plasmid structure.

Bacillus subtilis↗
PubMed1990

[Natural transformation in bacteria].

Transformants may be formed by some bacterial species when the growing cultures are mixed. This phenomenon caused by the DNA release from bacterial cells is called natural transformation. DNA release is most likely to be mediated by cell autolysis. Both chromosomal markers and plasmids are transferred by natural transformation. The phenomenon is reproduced while growing bacteria together in sterile soil. The DNA adsorbed on sand and other soil solid particles was more resistant to DNAse action, than the free transforming DNA. Natural transformation seems to be one of the forms of the genetic exchange in bacteria in their habitats. An indirect argument for this suggestion is perfect coordination between the different steps of transformation process, at least, in some bacterial species.

DNA, Bacterial↗
PubMed1989

[The effect of various rec-mutations on the frequency of the Tn917 transposition in Bacillus subtilis cells].

The effect of 11 rec-genes on the transposition frequency of Tn917 has been studied. Transposition frequencies in RecP, RecF15, RecB3 mutants differed from the ones in the control strains. The collection of mitomycin-sensitive mutants has been tested for transposition proficiency. The mms315 mutation decreasing the transposition frequency possess the properties of the rec mutation too.

Bacillus subtilis↗
PubMed1988

Interplasmidic illegitimate recombination in Bacillus subtilis.

The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1-3 X 10(-8). Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9-15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.

Bacillus subtilis↗
PubMed1988

[RNA synthesis during the stationary growth phase in the Bacillus subtilis Cgr4 mutant].

RNA synthesis was studied in Bacillus subtilis Cgr4 grown in the mineral sporulation medium enriched with glucose up to 2% and amino acids up to 1%. To study mRNA synthesis, a method of transfer of the 3H-uridine pulse-labeled culture to the supernatant of physiologically identical, not labeled culture, followed by further incubation was used, the amount of 3H-uridine in the supernatant as well as in cells being measured. RNA was also analysed electrophoretically and distribution of the label among the fractions was determined. It is shown that mRNA synthesized in the logarithmic phase degrades up to 12% on the 2nd hour of growth during 10 min; the mRNA in the stationary phase is stable on the 7th hour of growth; no degradation is observed in the course of 2-3 hours. The beginning of degradation coincides in time with secondary induction of the synthesis of serine proteases and with the onset of sharp decrease in incorporation of 3H-uridine in RNA as well as with induction of spore morphogenesis. On the basis of electrophoretical analysis of pulse-labeled RNA, it was demonstrated that, prior to the transfer, labeled uridine was included and preserved in RNA fraction for 2-3 hours after the transfer, this fraction corresponding in mobility with mRNA in polyacrylamide gel. The following conclusion may be drawn: stable mRNAs are synthesized in the stationary phase and may be used for the translation of extracellular serine protease.

Bacillus subtilis↗
PubMed1988

[Cloning in cells of Bacillus subtilis and study of the characteristics of promoter regions of DNA of Bacillus mesentericus and Phage SPO2].

11 fragments of Bacillus mesentericus and phage SPO1 DNA containing promoters were cloned in Bacillus subtilis. Nucleotide sequences of these fragments were determined and S1 mapping of transcriptional start points was performed. It was found that some fragments contained tandem or overlapping promoters. Nucleotide sequences of Bacillus mesentericus promoters were compared with known promoters from bacilli. Promoter activities were determined in different phases of cell growth. Nucleotide sequences of several promoters coincided with known sigma 43, sigma 29, sigma 28-promoters. Nucleotide sequences of other promoters differ greatly from those used by the known forms of Bacillus subtilis RNA polymerase. The existence of at least four other forms of bacilli RNA polymerase specific for these promoters is suggested.

Bacillus subtilis↗
PubMed1988

[Use of the thermoinducible temperate phage Phi 105cts139 for cloning in Bacillus subtilis].

The gene for alpha-amylase from Bacillus amyloliquefaciens having a foreign promoter providing gene expression in logarithmic growth phase and the cat gene of plasmid pC194 (AC fragment) were inserted into thermoinducible prophage phi 105 cts139. Possibility of amylolytic activity enhancement was studied after thermoinduction. When AC fragment and random PstI restricts of phage DNA were ligated and used to transform Bacillus subtilis 1A289 (phi 105 cts139) the Amy+ CmR transformants were obtained having the different levels of increased amylolytic activity (maximum--26 fold). Numerous phages without insert found in induced lysates suggest that insertions were unstable and (or) the clones were double lysogens for hybrid and original type phages. Stable insertion of AC fragment replacing the PstI-H-fragment of phage DNA revealed that all Amy+ CmR transformants were double lysogens. Inducibility depended on the insertion orientation.

Bacillus subtilis↗
PubMed1988

[Interplasmid illegitimate recombination in Bacillus subtilis cells].

The illegitimate recombination between S. aureus plasmids pE194 (or pGG20-the hybrid between pE194 and E. coli plasmid pBR322) and pBD17 (plasmid pUB110 without Hpa-II-C-fragment) in B. subtilis was studied. Plasmid cointegrates were generated with the frequency of 1-3.10(-8). Among the 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all the parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions has revealed that in 8 cases recombination occurred between short homologous regions (9-15 b.p.). One of the recombinants resulted from nonhomologous recombination. The similarity between nucleotide sequences of recombination sites of two types of contegrates and those used for pE194 integration into the B. subtilis chromosome (Bashkirov et al. 1987) was demonstrated. Possible mechanisms of illegitimate recombination are discussed.

Bacillus subtilis↗
PubMed1987

Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions.

The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.

Bacillus subtilis↗
PubMed1987

Various means of integration of the expressible human dihydrofolate reductase gene into the Bacillus subtilis genome.

Integration of expressible DNA corresponding to the human dihydrofolate reductase (hDHFR) gene into the Bacillus subtilis genome has been achieved in different ways. The clones obtained contained one to seven copies of this gene per genome equivalent and were resistant to trimethoprim. Clones produce a new protein coded by the integrated hDHFR gene. In all clones, the integrated DNA was stably maintained even under nonselective growth conditions.

Bacillus subtilis↗
PubMed1987

[Insertion mutagenesis in Bacillus subtilis (Marmur)].

A method for obtaining mutations in Bacillus subtilis using integration into the chromosome of the plasmid pHV60 ligated to chromosomal DNA fragments was developed. Auxotrophic mutants acquire, in addition, chloramphenicol-resistance, due to insertion of appropriate plasmid determinants. Chromosomal localization was established and the properties of several mutants were studied.

Bacillus subtilis↗