DNA polymorphism of dogs (Canis familiaris L.). III. VNTR- and STR-loci. Their use in dog breeding and in criminalistics.

The application of mini- and microsatellite polymorphisms of dog DNA, also referred to as VNTR- and STR-loci, respectively, in dog breeding and criminalistics is considered. Their use in dog breeding is shown to clarify pedigrees, establish paternity and purebred, as well as to differentiate breeds mainly in the form of microsatellite DNA polymorphism. In criminalistics, dogs can be both participants in crime scenes in the form of attacks on humans or pets, and some witnesses through whose DNA extracted from their fur or feces, by DNA identification of a particular dog, it may be help to get out to the perpetrator or at least to the crime scene, which is also a good help in its disclosure. At the same time, population studies of dogs, during which the prevalence of certain alleles of marker traits in the form of STR loci in different territories is established, contribute to making the right decisions. The databases on STR-polymorphism of dog DNA or their prototypes are briefly described. Attention is paid to the sources of forensic canine DNA, as well as methods of its extraction and preliminary evaluation of isolated preparations. The use of VNTR polymorphism was rather short-lived, and was quickly replaced by STR polymorphism. There are some trends in the introduction of new polymorphic traits in this area in the form of single-nucleotide polymorphism or SNPs, potentially providing more accurate information, including for DNA identification of individuals. The issues of universal DNA certification of dogs are discussed, which can contribute to improving the culture of keeping dogs and will allow the elimination of stray dogs in the future, which will be humanistic character and potentially reduce the number of aggressor dogs.

Salt stress influence on stomatal and hydraulic conductivity, as well as on the level of aquaporins in leaf cells of barley plants, differing in salt tolerance

We studied participation of aquaporins in the regulation of leaf hydraulic conductivity and relationship of hydraulic conductivity with accumulation of ABA and stomatal closure during salt stress. Using the method of immunohistochemical localization we showed that salinity led to greater decline in the level of aquaporins in the region of the vascular leaf bundles of the more salt-tolerant Prairia cultivar, accompanied by a noticeable decrease in hydraulic conductivity of the leaf. In the less salt-tolerant plants of the Mikhailovsky cultivar, significant changes in the level of aquaporins under the influence of salt stress were not found. The degree of decrease in the hydraulic conductivity of the leaf in plants of two cultivars under the influence of salt stress correlated with a decrease in transpiration. Immunohistochemical localization of abscisic acid (ABA) in leaf cells showed that during salt stress this hormone accumulated in leaf mesophyll cells and stomata. The uptake of exogenous hormone from the nutrient solution and its entry into the leaf through the vascular bundles was accompanied by an increase in staining for aquaporins and the hydraulic conductivity of the leaves, which is characteristic of the ABA action. Differences in the localization of exogenous and endogenous hormones were obviously the cause of the opposite directions of changes in hydraulic conductivity: its increase under the influence of an exogenous ABA and a decrease - under the influence of salt stress. ABA concentration assessment in xylem showed the absence of its increase during salt stress, which explains the absence changes of staining for this hormone in the region of the leaf vascular bundles and indicates that accumulation of ABA in a short-term salt stress is not the result of its delivery from the roots, but the result of its synthesis in the shoot itself.

Contribution of the TROLC gene to the regulation of tobacco growth in response to stress factors.

The trolC gene refers to plast genes that have entered the genome of Nicotiana tabacum as a probable result of horizontal transfer from Agrobacterium rhizogenes. It was shown that the trolC gene is expressed in young tissues of wild type tobacco; however, the physiological functions of the product of this gene remain largely unknown. The aim of our work was to obtain transgenic tobacco plants expressing a fragment of the trolC gene under the control of the 35SCaMV promoter in an antisense orientation and to assess the growth parameters of their roots under the action of abiotic stress factors. For morphometric analysis, 8 lines of transgenic plants were used. The analysis of root growth under the action of sodium chloride (100 mM), cadmium acetate (100 μM) and hypothermia (12°C) was conducted. Transgenic plants were characterized by improved shoot growth parameters under normal conditions. The roots of transgenic plants grew more slowly under normal conditions and under the action of cadmium and hypothermia than in wild type plants. The product of trolC gene has a negative effect on shoot growth, a positive effect on root growth, and also participates in the regulation and maintenance of root growth under the action of cadmium and hypothermia.

DNA polymorphism of dogs (Canis familiaris L.). I. Origin, distribution of dogs in the light of molecular biological data about their mitochondrial and nuclear genomes

Information is given on the size and organization of the mitochondrial and nuclear genomes of dogs, including information on polymorphisms of some loci used to clarify the phylogenetic relationships of wolves and the first dogs, including hypotheses about the places of domestication of ancient now extinct wolves and the dates of these events. It is noted that the introduction of molecular biological methods in archaeology has allowed to obtain the principally new data on ancient wolves and dogs. Based on mtDNA polymorphism and nuclear DNA polymorphism, migration routes of already domesticated dogs together with humans have been tracked. The previously existing points of view about the origin of the first dogs in Western Europe, as well as in East Asia, have been supplemented in recent years by assumptions about the appearance of the first proto-dogs in Siberia.

Comparative analysis of the effect of epibrassinolide and 6-benzylaminopurine on the key components of the glutathione complex in the roots of wheat seedling under salinity.

The study carried out a comparative analysis of the effect of 24 epibrassinolide (EB) and 6-benzylaminopurine (BAP) on the growth and state of the main components of the glutathione system in the roots of wheat seedlings under the action of 2% NaCl, which for the first time revealed the ability of these phytohormones to similarly stabilize stress-induced decrease GSH / GSSG ratio, positively regulate the activity of glutathione reductase and glutathione-S-transferase. A comparable level of protective effect of BAP and EB on root length was revealed. The data obtained indicate that endogenous cytokinins may play the role of hormonal intermediates in the implementation of the protective.

DNA polymorphism of dogs (Canis familiaris L.). II. RAPD-analysis.

A rather simple method of detecting DNA polymorphism in dogs in the form of amplification of random fragments of the genome using RAPD analysis with primers with arbitrary nucleotide sequences, which does not require prior knowledge of the sequences of nitrogenous bases of the detected DNA fragments, is considered. However, information about the complete genomes of the investigating species of organisms and, dogs in particular, allows with a preliminary in silico RAPD analysis to predict the expected results for each primer and their multiplex composition, which makes it possible at this stage to reject unsuitable primers before conducting "wet" experiments. In the case of known genomes of a number of related organisms for example in this case dogs, the multiplex RAPD analysis conducted in silico with six decameric primers, the sequences of which exclude the formation of homo- and heterodimers in PCR showed both genomic similarities and differences between different breeds, as well as with their wild relative gray wolf. Multiplex in silico RAPD analysis is a virtual PCR and if amplicons common to all studied organisms are removed from such amplification, leaving only significant ones, then this tool in the form of the ABCDNA_GS program created by us can be used to establish phylogenetic kinship, especially of those organisms whose genomes are highly collinear, which is just typical of the genomes of different dog breeds.

Tracing in hydrogeology of underground watercourses using DNA tracers

Underground water sources and their movement in various rocks, including karsts, are of considerable interest for many reasons, among which human economic activity plays a significant role. Various compounds are used to trace underground waters – stable and radioactive isotopes, microorganisms and bacteriophages, solid particles as lycopodium spores, chlorides and bromides, other ions, as well as fluorescent dyes. Relatively recently, DNA molecules have been used for this purpose, which can be detected with high sensitivity using a polymerase chain reaction. The commonly used number of DNA tracers is 1015 - 1016 molecules, which, despite significant dilution by water flows, ensures their subsequent detection. In addition to the high sensitivity of such analyses, other advantages of DNA molecules for tracing watercourses are their harmlessness for ecology, relative cheapness. In view of the possible destruction of DNA molecules in an aqueous medium and their sorption on various rocks, it is proposed to use encapsulated molecules, the shells of which are polylactic acid, silica, alginate and other compounds. An experiment on tracing the watercourse of the Tyutyulena stream from the ponor at the Kosh cave to the Traval griffin is predicted, indicating that in this case, taking into account the water flow, only about 1012 DNA molecules will be enough.

Bacterization of forage grass seeds: Influence on spreading and growth of plants.

Inoculation of seeds before sowing of growth-stimulating microorganisms is an effective method for increasing the productivity of crops and forage grasses, in particular. In this work, nine strains of bacteria were tested for their ability to produce the phytohormone indolyl-3-acetic acid (IAA) and stimulate seed germination and elongation of shoots and roots of seedlings of mogar, sudanese grass, awnless brome, alfalfa, and meadow fescue. The highest IAA concentration in the culture liquid was found in the strains Enterobacter ludwigii BLK and Pseudomonas sp. UOM 10 (2001 and 1617 ng/ml, respectively), and the lowest was in Pseudomonas sp. 5GCH and P. koreensis IB-4 (144 and 141 ng/ml, respectively), however, at this stage of research, a direct relationship between the level of phytohormone synthesis and the ability of bacteria to positively affect plant growth was not found. The plants were characterized by the species specificity of the reaction to bacterial treatment, with the exception of the Pseudomonas sp. 5GCH, which in some cases had a positive effect on seed germination, but did not enhance the growth of roots and shoots in the studied herbs. Synchronous stimulation of seed germination and an increase in the length of shoots and roots was observed on mogar plants upon inoculation with E. ludwigii BLK and P. plecoglossicida CH5-2 and on an awnless brome (P. chlororaphis 4CH, B. vietnamiensis 8CH, E. ludwigii BLK, P. laurentiana ANT 17 and P. plecoglossicida CH5-2). None of the strains simultaneously increased the length of shoots and roots in Sudanese grass and meadow fescue, but most microorganisms enhanced seed germination in them. Bacterization did not increase germination in alfalfa, and only Pseudomonas sp. UOM 10 and P. koreensis IB-4 promoted the growth of its shoots and roots.

Genetic engineering of cyclomaltodextrin glucanotransferases

Studies of cyclic oligosaccharides from six, seven and eight glucose residues, designated as alpha-, beta- and gamma-cyclodextrins, respectively, and everything related to them have been going on for 130 years. In this review, the history of the study of these molecules is briefly considered. The interest in cyclodextrins is caused by their ability to form inclusion complexes with a number of organic and inorganic compounds, radically changing some of their properties. This is widely used in the pharmaceutical, cosmetic and food industries, and beta-cyclodextrin is even registered as a food additive E459. Cyclodextrins are obtained from starch under the action of cyclodextringlucanotransferase (CGTase) enzymes, a characteristic feature of which is their non-strict specificity in relation to the types of oligosaccharides produced. The main producers of these enzymes are a group of bacteria of the order Bacillales, which unites several families (Paenibacillaceae, Bacillaceae, Thermoactynomicetaceae, etc.), but in last years CGTases have been found in a wide range of bacteria and archaea. The genetic engineering of CGTases began in the middle of 1980s, after the CGTase gene from Paenibacillus macerans (formerly Bacillus macerans) was cloned and sequenced for the first time, and during this period rather noticeable progress was made in understanding the organization and functioning of these enzymes, including using X-ray diffraction analysis. With the help of site-directed mutagenesis, error-prone PCR, as well as by creating chimeric forms of these enzymes, certain successes have been achieved in recent decades in changing (improving) the specificity of their action. Suitable leader peptides are used to increase the synthesis and secretion of genetically engineered CGTases, and various heterologous producers are also proposed, including the bacteria Escherichia coli, B.subtilis, Lactococcus lactis and the methylotrophic yeast Koagataella phaffii.

LAMP amplification of nucleic acids. I. Two decades of development and improvement.

Over the two decades that have passed since the development of loop amplification (Loop AMPlification, LAMP), that carry out to detect specific nucleic acid under isothermal conditions, it has undergone quite a lot of improvements. This review presents data represented methodological bases of about a hundred variations of the LAMP, classified according to the methods of detecting both target DNA products (lamplicons) and by-products (pyrophosphate and protons), taking into account the specificity of the processes, and according to the purpose of certain LAMP options and implementation, including microfluidics. Particular attention is paid to quantitative LAMP amplification and promising digital LAMP. The prospects for the development of the method are presented.