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V G Debabov

Publications and source records attributed to V G Debabov.

At least 55 records · Page 3Linked to original sources
PubMed1988

[Molecular cloning and the expression of the genes of amino acid biosynthesis of Corynebacterium glutamicum in Escherichia coli cells].

Cloning of genes for threonine and lysine biosynthesis from Corynebacterium glutamicum was performed in Escherichia coli cells using the plasmid vector lambda pSL5. The cloned genes are identified via complementation of thrB and lysA mutations. The gene complementing thrB of E. coli is located within a 4.1 kb EcoRI fragment of C. glutamicum chromosomal DNA. All the recombinant phasmids complementing the lysA gene of E. coli contain common 2.2 kb and 3.3 kb EcoRI C. glutamicum DNA fragments. The cloned DNA fragments hybridize with identical EcoRI fragments of C. glutamicum chromosomal DNA.

Amino Acids↗
PubMed1987

[Construction of derivatives of the diphtheria toxin gene and their expression in Escherichia coli cells].

A fragment of diphtheria toxin (tox) gene from beta 45 phage DNA was cloned on pUC19 plasmid in E. coli cells. The fragment is coding for toxA fragment of the toxin and contains the control region of the tox gene. The tox gene promoter is active in E. coli. The toxA protein is found mainly in periplasm of E. coli cells. The protein is enzymatically active in ADP-ribosilation of elongation factor 2 from eucaryotic cells. An in frame toxA-lacZ' fusion was constructed on pUC8 plasmid. The hybrid protein expresses both toxA and lacZ' activities. Two or seven base pairs were deleted from the central part of toxA gene by means of S1 nuclease digestion. Translation of hybrid toxA-lacZ' mRNA should be terminated downward the delections due to the frameshifts caused by them. Nevertheless, a functionally active alpha-peptide of beta-galactosidase is expressed by both the deletion fusions. The existence of another translational start site functioning in E. coli and located inside 3'-end region of toxA mRNA is suggested.

Base Sequence↗
PubMed1987

[Advances in gene engineering of microorganisms].

A novel branch of national economy--biotechnology is being developed, based on genetic engineering. The construction of strains using the methods of molecular cloning has led to-date to creation of new biotechnological processes. Further advance in biotechnology would be mainly promoted by the possibilities of application of gene engineering to reorganization of industrially important microorganisms. These are bacilli employed for production of vitamins, enzymes, insecticides; streptomycetes--the producers of antibiotics; yeasts applied in bakery industry, in production of fodder proteins; pseudomonads which will be helpful in development of effective biological means for protection of environment, etc. So, vector molecules based on plasmids and phages have been constructed for best-studied representatives of industrial microorganisms, the methods of introduction into the cell of hybrid DNA molecules worked out, the problems of optimization of foreign gene expression being currently solved.

Bacteria↗
PubMed1987

[Cloning of the regulator gene of Erwinia carotovora repressing pectate lyase ptlA gene expression].

Pectate lyase synthesis in the cells of Erwinia carotovora ELA 49 is induced by polypectate. This suggested that the Erwinia chromosomes carried a regulator gene responsible for negative regulation of the pectate lyase gene expression. In the present study the regulator gene controlling expression of one of the pectate lyase structural genes was cloned and designated as ptlA gene. For this purpose a genetic system with the tester plasmid pPc624 as the main element was constructed. The tester plasmid contained cat gene (resistance to chloramphenicol) controlled by the promotor of the ptlA gene cloned on vector pPD620. Plasmid pPC624 was maintained in the E. coli cells in a number of 1-2 copies and transferred resistance to chloramphenicol in concentrations up to 100 micrograms/ml to the cells. The E. carotovora cells containing pPC624 were sensitive to chloramphenicol in media containing no inductor (sodium polypectate). In media with the inductor they were resistant to chloramphenicol. Therefore, plasmid pPC624 proved to be a suitable system for testing the regulator gene product. The E. coli cells containing plasmid pPC624 were transformed by the hybrid Ptl+ plasmids identified in the clonotheque of the Erwinia DNA EcoRI fragments. The E. coli cotransformants were characterized by chloramphenicol sensitivity which provided a conclusion that the regulator ptlR gene controlling the ptlA gene expression was localized on the DNA EcoRI fragment (7.3 kb) containing the pectate lyase ptlA and ptlB genes. Deletion analysis showed that the investigated genes were localized in the EcoRI fragment (7.3 kb) of the E. carotovora chromosomal DNA in the following order: ptlA--ptlB--ptlR.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloramphenicol↗
PubMed1982

Nucleotide sequence analysis of the cloned salmon preproinsulin cDNA.

A cDNA library was constructed using polyadenylated RNA from salmon (Oncorhynchus keta) Brockmann bodies, plasmid vector pBR322, and in vitro recombinant DNA techniques. Insulin-related clones were identified with a cDNA probe generated from the same RNA and enriched for insulin sequences. Two recombinants were shown to contain the nucleotide sequence of the entire coding region and parts of the 5' and 3' untranslated regions. The salmon preproinsulin mRNA is about 760 nucleotides long, 315 of which code for the protein, while about 190 and 200 nucleotides belong to the 5' and 3' flanking regions, respectively. Comparison of the nucleotide sequences of salmon insulin mRNA with those from other species reveals that sequence conservation is limited to the regions coding for the B and A peptides and two segments of the signal peptide. The C-peptide region exhibits no significant sequence homology with the C-peptides of other vertebrates. The 5' and 3' untranslated regions of the salmon preproinsulin mRNA are homologous only with the anglerfish mRNA, whereas there is no evident homology with those of birds and mammals. In addition to establishing the sequence of the preproinsulin mRNA, cloned salmon insulin cDNA provides a specific probe for the analysis and isolation of genomic DNA fragments containing insulin genes.

Amino Acid Sequence↗
PubMed1982

[Instability study of Escherichia coli strains containing hybrid plasmids with threonine operon fragments].

The stability of Escherichia coli strains carrying hybrid plasmids which contain ColE1-like replicon and threonine operon genes was studied. It was shown that the main reason for instability is the loss of a plasmid. The second reason for instability is the rec-dependent recombination that leads to formation of new plasmids. All experiments where instability of strains was observed, can be quantitatively described by the model that presumes a random loss of plasmids in cell population with a frequency of about 7.10(-4), which results in diappearance of plasmid-borne cells due to their low growth rate. Instability increases during the stationary phase but it is not easy to quantitatively estimate this process.

Cloning, Molecular↗
PubMed1977

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

A convenient procedure for the isolation of specific EcoRI-fragments of E. coli genome and their amplification on Km-resistance plasmid victor CK delta11 is described. The hybrid molecules were constructued in vitro using EcoRI-digestion, followed by ligation. Then appropriated E. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK delta11-arg+, CK delta11-his+, CK delta11-thr+ and CK delta11-leu+ containing loci of E. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of one EcoRI-fragment of initial plasmid CK delta11 and one respective specific portion of E. coli genome.

Arginine↗
PubMed1977

[Isolation and preliminary characteristics of the extrachromosomal elements of Bacillus thuringiensis DNA].

Centrifugation of Bacillus thuringiensis 351 DNA in CsCl-ethidium bromide density gradient has revealed, besides the main band of chromosomal DNA (p = 1.56 g/cm2), some additional bands with higher density, which usually correspond cicle superhelix DNAs, are revealed. 6 discrete bands are observed under electrophoresis of total fraction of minor DNA bands, which suggests, that the preparation contains cicle DNAs of different size. The treatment with DNAse results in the appearance of 3 bands corresponding to opened cicle DNA forms, their molecular weight being 12-10(6)--4-10(6) daltons. Cicle super-helix and opened cicle DNA forms are found in minor fraction by means of electrone microscopy. Statistical analysis has revealed the presence of at least three types of cicle molecules of different size. The variant 351-10, free of extrachromosomal elements, is isolated after ethidium bromide treatment at high temperature. Possible mechanisms of cicle DNAs formation are discussed. Biological function of DNA extrachromosomes fragments in Bacillus thuringiensis is still obscure.

Bacillus thuringiensis↗
PubMed1977

[Effect of EcoRI restrictase on the transforming DNA of different bacilli].

The transforming activity of DNA from Bacillus subtilis 168, Bac. subtilis W23, Bac. subtilis NRS and Bac. aterrimus after the EcoRI restrictase treatment was studied. The auxotrophic strains of Bac. subtilis 168 were used as recipients in bacterial transformation. The transforming activity for different markers decreased up to 0.001--6 per cent from the initial level for different Bacilli DNAs. In some cases the sensitivity of the same marker from different Bacilli differed in more than 50 times. Bac. subtilis and Bac. aterrimus have demonstrated the maximal differences. Such differences can be used for the identification of close related Bacilli.

Bacillus subtilis↗
PubMed1976

[Transforming activity of Escherichia coli DNA fragments obtained following endonucleolytic splitting by EcoRI restrictase].

Escherichia coli DNA with molecular weight of 20 - 10(6) daltons was digested by restriction endonuclease EcoR1, and the transforming activity of resctricts was studied. The transforming activity of restricts for two markers (Leu and Arg) was 2-5 fold increased, for two other markers (Thr and His) was not changed, and for one marker (Pro) was completely absent. The molecular weight of E. coli DNA restricts was 7,5-10(6) daltons. An increase and a decrease of the transforming activity for different markers appeared to be the result of two effects: 1) more efficient uptake of low molecular weight DNA into the cell and 2) the inactivation of markers as a result of location close to EcoR1 induced break.

Catalysis↗
PubMed1976

[Simple method for isolation of three DNA-polymerases from E. coli using chromatography on DNA-cellulose].

A simple method for isolation of three DNA-polymerases from E. coli is developed; it is based on properties of the enzymes to bind with single-stranded DNA, fixed on cellulose. DNA-polymerases I, II and III were identified by their relation to templates, ionic strength, heat denaturation and inhibition of the activity with agents which blocked sulfhydryl groups. After purification which included chromatography on DNA-cellulose as well as fractionation with phosphocellulose and gel-filtration through Sephadex G-100, the enzymes were free the endonucleases contamination.

DNA Polymerase I↗