Biomedical subjects
Publications and source records attributed to Iu I Kozlov.
After inactivation of the ypaA gene in Bacillus subtilis, the phenotypic pattern obtained showed that this gene controls a system for active flavin transport and, possibly, riboflavin excretion under the conditions of constitutive synthesis.
The PCR copy of the ribR gene of Bacillus subtilis was subcloned in Escherichia coli cells under the control of the phage T7 inducible promoter. The polypeptide of 26 kDa corresponding to the 690-bp gene is the product of the ribR gene. The protein encoded by the ribR gene is flavokinase, and the riboflavin-reduced form is the substrate for it.
A clinical example of effective treatment of an adult patient with combined deformations of the jaws (upper prognathism of the third degree and open occlusion of the second degree) is reported. Fragmented osteotomy of the maxilla at the level of symmetrical removal of second molars is performed. Due to Engle's arches soldered with Vasil'ev's splint and compression, a maxillary fragment was shifted 10 mm backward and downward at the expense of compressing the inter-root septae; the first molars were preserved. Diastemata and tremata between incisors of both jaws were eliminated by the same method.
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Assessment of specific activity of riboflavin synthase and the level of riboflavin accumulation in strains with a 110-nucleotide deletion in the regulatory region of the riboflavin operon showed that this deletion specified semi-constitutive expression of the operon. This was assumed to be connected with the elimination of three nucleotides from a potential transcription antiterminator.
A 13.0-kb EcoRI fragment of Bacillus subtilis DNA carrying an additional regulatory ribR gene of riboflavin operon was cloned on the basis of resistance to 7, 8-dimethyl-10 (O-methylacetoxym)-isoalloxasin. The cloned fragment was trans-dominant with regard to ribC constitutive mutations that block the overproduction of riboflavin but inactive relative to constitutive mutations in the rib(O) regulatory region.
The sequencing of a 3.5 kb EcoRI-BamHI fragment located in the 236 degrees region of the Bacillus subtilis chromosome revealed a 690-bp long open reading frame, partly similar in amino acid composition to flavokinases/FAD synthases from several microorganisms. The discovered sequence can be identified with the gene ribR, an auxiliary regulatory gene of the riboflavin operon of Bacillus subtilis.
The authors have generalized the experience of treatment of 82 injured with injuries of condylar processus fracture treatment of the lower jaw. By a surgical way were successfully eliminated posttraumatic ankylosis and contracture of temporomandibular articulations with use of titanium endoprosthesis and semi-joints. The osteosynthesis of condylar processus of the lower jaw by titanium miniplates and screws was also practiced.
Four types of protease negative mutants of Bacillus amyloliquefaciens A50 were selected after four stages of step-by-step UV light mutagenesis. EDTA, and PMSF were used as inhibitors of protease activity to characterize the protease negative mutants with regard to the protease type. The electrophoretic patterns of proteases from culture medium of B. amyloliquefaciens protease deficient mutants were studied. The proinsulin stability in the culture medium of different mutants was analysed. Protease deficient B. amyloliquefaciens strain A50-32 may be used as a recipient for cloning and expression of a foreign proteins genes.
Recombinant strains of S. lividans capable of secreting streptavidin were isolated. Various constructions containing either streptavidin gene copies integrated within a chromosome or a streptavidin gene within the secretory vector were investigated. S. lividans SA2 containing a gene copy integrated within the chromosome had the maximum productivity amounting to 60-80 per cent of the S. avidinii productivity. New vectors for Streptomyces used in the study are described.
Streptococcal broad host range plasmid pAM beta 1 was transferred by a conjugation-like process from Streptococcus faecalis to 13 strains of different Bacilli species. In intraspecies matings the frequencies of transfer of pAM beta 1 varied from 2.10(-5) to 1.10(-8). As it was shown by comparative analysis the frequency of transfer and stability of the maintainance of plasmid pAM beta 1 in Bacilli were not connected. Molecular weight and restriction pattern of pAM beta 1 DNA isolated from Bacilli were the same as those of pAM beta 1 DNA from Streptococcal donor strain.
The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.
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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.
A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid. This fragment has been mapped using some restriction endonucleases. Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids. Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h. When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated. For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype. It is possible that the great amount of mutant enzyme molecules compensated their low activity. On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.
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A set of hybrid plasmids carrying Escherichia coli threonine genes was obtained and cloned. The plasmid pBR322 was used as a vehicle. The genetic and restriction analyses showed that genes thrA and thrB were placed between SalGI and EcoRI sites on the 2.6 megadaltons DNA region. The transcription of threonine operon genes inserted in the hybrid plasmids is under the control of its own promoter. The copy number of hybrid plasmids was reverse proportional to their molecular weight and did not depend on the replicon number. Amplification of genes of threonine operon by hybrid plasmids led to 20-25-fold increase of homoserine dehydrogenase activity, encoded by thrA gene. The expression of this gene, incorporated in hybrid plasmids, was repressed by the addition of threonine and isoleucine in the culture medium.