A novel approach for Escherichia coli genome editing combining in vivo cloning and targeted long-length chromosomal insertion.

Despite the abundance of genetic manipulation approaches, particularly for Escherichia coli, new techniques and increased flexibility in the application of existing techniques are required to address novel aims. The most widely used approaches for chromosome editing are based on bacteriophage site-specific and λRed/RecET-mediated homologous recombination. In the present study, these techniques were combined to develop a novel approach for in vivo cloning and targeted long-length chromosomal insertion. This approach permits direct λRed-mediated cloning of DNA fragment with lengths of 10kb or greater from the E. coli chromosome into the plasmid vector pGL2, which carries the ori of pSC101, the ϕ80-attP site of ϕ80 phage, and an excisable CmR marker bracketed by λ-attL/attR sites. In pGL2-based recombinant plasmids, the origin of replication can be eliminated in vitro via hydrolysis by SceI endonuclease and recircularization by DNA ligase. The resulting ori-less circular recombinant DNA can be used for targeted insertion of the cloned sequence into the chromosome at a selected site via ϕ80 phage-specific integrase-mediated recombination using the Dual-In/Out approach (Minaeva et al., 2008). At the final stage of chromosomal editing, the CmR-marker can be excised from the chromosome due to expression of the λint/xis genes. Notably, the desired fragment can be inserted as multiple copies in the chromosome by combining insertions at different sites in one strain using the P1 general transduction technique (Moore, 2011). The developed approach is useful for the construction of plasmidless, markerless recombinant strains for fundamental and industrial purposes.

[OPTIMIZATION OF REPARATIVE OSTEOGENESIS JAWS ON AGING (PRECLINICAL STUDIES)].

The article presents the results of pre-clinical studies of the possibility of optimization of reparative osteogenesis in postoperative bone cavities of the jaws in old rats with a peptide bioregulator of Kartalaks. Using histological and morphometric methods in the experimental study, it is shown that the use of Kartalaks positive effect on the course of the process of reparative osteogenesis of the mandible in old rats. The features of the Kartalaks drug investigated for optimization of reparative osteogenesis jaw during aging. It is found that by using Kartalaks in old animals to 30 days before the surgical intervention, the process of reparative osteogenesis standard defect of mandible occurs at early stages (before 30 days), similar to the control. The deadlines for 60-120 days from the beginning of the experiment using the peptide bioregulator Kartalaks, noted the formation of a complex regenerate tissue containing a smaller proportion of connective tissue and cartilage, as well as more advanced remodeling of newly formed bone tissue compared with the control group, and older animals who received the drug after performing surgery.

[Gerontostomatology's topical issues in Russia at the present stage].

Gerontostomatology is one of the most important interdisciplinary sections of the medicine, studying mechanisms of aging of the chewing device of a person, and also carrying-out search of decisions in treatment of stomatologic diseases in persons of the senior age groups, at the same time increasing quality of their life. The article covers actual problems of modern gerontostomatology in Russia.

[Medico-social and psychosomatic status of gerontostomatologic patients].

Assessment of medico-social and psychosomatic status of patients of the advanced and senile age having dental diseases was carried out. The results of the study show that the level of stomatological morbidity in this contingent of patients is rather high, and the treatment must be carried out with obligatory participation of doctors-interns and taking into account mental health of a person.

[Application of bioregulating therapy in complex treatment of temporomandibular joint diseases in people of elderly and senile age].

The results of dynamic supervision over application a peptide bioregulator of "Sigumir" in complex treatment of 62 patients of the senior age groups with various diseases of a temporomandibular joint are presented. It is shown that application of a complex of treatment-and-prophylactic actions, including rational tooth prosthetics, functional and pharmacotherapy (including accompanying diseases), and also use a peptide bioregulator of cartilaginous and bone tissues "Sigumir" in complex therapy of patients with diseases of a temporomandibular joint of a various etiology, enables to stop in short-terms a painful syndrome in such patients, to increase amplitude of opening of a mouth, to improve chewing function, to reduce terms of treatment and to provide preventive maintenance of relapses of TMJ's pathology during all period of supervision.

[Detection of temporomandibular joint diseases in elderly and senile age in ambulatory medicoprophylactic institutions].

The research analyzes diagnostic TMJ's disease's identification work of stomalogists owned to different types of day care properties (municipal, departmental, private). This research based on examination of primary medical records of 1906 patients aged 61 to 89 years, including 2978 prescribing lists made by stomatologists - orthopedists to dental outpatient's card. This research shows that in case of outpatient conditions in the primary examination of patients of elderly and senile age stomatologists pay attention to joint pathology in the presence of acute patients' complaints, caused by TMJ's displacement, arthritis, painful TMJ's dysfunction or pronounced sound phenomena of TMJ. Stomatologists examine TMJ's pathology more particularly. The TMJ's pathology complicates the tooth replacement made to patients contrary to absence of indications of disease presence in primary medical records. The prepared conclusion and recommendations allow improving the outpatient diagnosis of TMJ's pathology.

Genetic control of the resistance to phage C1 of Escherichia coli K-12.

Escherichia coli K-12 lytic phage C1 was earlier isolated in our laboratory. Its adsorption is controlled by at least three bacterial genes: dcrA, dcrB, and btuB. Our results provide evidence that the dcrA gene located at 60 min on the E. coli genetic map is identical to the sdaC gene. This gene product is an inner membrane protein recently identified as a putative specific serine transporter. The dcrB gene, located at 76.5 min, encodes a 20-kDa processed periplasmic protein, as determined by maxicell analysis, and corresponds to a recently determined open reading frame with a previously unknown function. The btuB gene product is known to be an outer membrane receptor protein responsible for adsorption of BF23 phage and vitamin B12 uptake. According to our data the DcrA and DcrB proteins are not involved in these processes. However, the DcrA protein probably participates in some cell division steps.

[Cloning and complementation analysis of the Escherichia coli gpr locus, influencing DNA replication of certain lamdoid phages]. / Klonirovanie i komplemntatsionnyi analiz lokusa gpr Escherichia coli, bliiaiushchego na replikatsiiu DNK nekotorykh liambdoidnykh fagov.

Escherichia coli DNA fragments which suppressed gpr27 and gpr2 mutations in the earlier proposed gprA and gprB genes, respectively, were cloned within phage vectors. Mutations gpr2 and gpr27 restrict DNA replication of some lambdoid phages and are located in the region of dnaK, J genes. DNA-DNA hybridization showed that the cloned fragments correspond to the region where gpr mutations were genetically mapped and, in some cases, do not include dnaK, J genes. These results provide the evidence that gprA and gprB genes may be physically separated from dnaK, J genes.