Том 61, № 8 (2025)

One of the challenges in de novo DNA synthesis is the occurrence of errors. The number of errors associated with synthesis protocols limits the efficiency of obtaining synthetic DNA fragments, both on small and large scales. In this study, we propose a new rapid protocol for the synthesis of DNA fragments using the thermostable endonuclease V from Thermotoga maritima (Tma endonuclease) as an error-correction enzyme. The aim of the research is to develop a one-step error-correction method for de novo gene synthesis. The recombinant Tma endonuclease enzyme was obtained. To evaluate the efficiency of error correction using Tma endonuclease, the synthesis of the mTomato gene was performed using various protocols. The enzyme's efficiency was assessed visually based on the number of fluorescent colonies. To confirm the results, random samples of colonies were selected, and the assembled sequences were sequenced using the Sanger method. It was demonstrated that Tma endonuclease can be used in both the classical three-step and the rapid one-step error-correction protocols without loss of activity. It was also established that, with comparable accuracy of DNA fragment synthesis, the efficiency of Tma endonuclease is similar to that of the commercial enzyme Correctase (ThermoFisher). It was concluded that one-step error correction using endonuclease V accelerates the process of DNA fragment synthesis and error correction. By reducing the number of steps, this protocol is better suited for high-throughput DNA synthesis platforms compared to the classical protocol using non-thermostable enzymes.

One of the central problems in molecular biology is to establish the mechanism of DNA-protein recognition. A promising model for elucidating the subtle processes of recognition is to study individual stages occurring during specific interaction of the regulatory protein with the operator. Determination of the structure of DNA and nucleotides involved in the interaction of the operator with the repressor allows us to elucidate the pathways of transcriptional regulation of gene expression. Analysis of the regulatory mechanisms of expression of genes encoding bacterial toxins allows us to understand the options for suppressing the toxin genes expression, which will ultimately ensure the creation of a system of regulated synthesis of bacterial toxins. Being study the role of the spatial structure and individual nucleotides of the operator region of the Bacillus cereus hlyII gene in the interaction with the transcriptional regulator HlyIIR, the regions of the operator region of hlyII gene that are essential for the interaction with the HlyIIR repressor were determined. The efficiency of the specific interaction of the HlyIIR repressor with its operator was determined depending on its DNA spatial structure. Using synthetic oligonucleotides with substitutions of individual nucleotides specific nucleotides of the operator were identified that ensure effective specific interaction with the repressor. Thus, changes in the profiles of the efficiency of DNA-protein recognition depending on the structure of the operator were shown.

Using DNA markers, homozygous plants with combinations of Pp-D1 and Pp3 genes exhibiting purple pericarp, as well as the Pp-D1, Pp3 and Ba1 genes resulting in black grain color of due to anthocyanins accumulation in pericarp and aleurone layer were isolated from six hybrid F₂ populations obtained with participation of spring soft wheat varieties: Novosibirskaya 31, Sibirskaya 21 and Leader 80. Hybrid plants with a high level of anthocyanins, phenolic compounds, and antioxidant activity were isolated; these can be used to obtain improved commercial soft wheat varieties with increased content of biologically active components in grain, adapted to the conditions of Western Siberia. It was shown that the dominant allele Pp-D1 was found in 20% of Russian varieties and in 15 of 37 analyzed varieties of European selection, which will allow for the application of a simplified scheme for obtaining new hybrids with purple pericarp by controlling only the transfer of the Pp3 gene.

The intragenomic polymorphism of the OCA2 gene fragment (approximately 4800 bp), encoding the transmembrane P-protein associated with feather coloration in birds, was studied for the first time in seven Far Eastern owl species: Eurasian eagle-owl (Bubo bubo), Blakiston's fish owl (Bubo (Ketupa) blakistoni), snowy owl (Bubo (Nyctea) scandiacus), long-eared owl (Asio otus), Ural owl (Strix uralensis), oriental scops owl (Otus sunia), and Japanese scops owl (Otus semitorques). The intragenomic variability of OCA2 within this diverse group of Strigiformes ranged from 0.005 in B. bubo to 0.014 in O. sunia, exceeding interspecific values for this gene in the genera Falco and Cygnus (0.000–0.006). Genetic distances between genera within the family Strigidae varied widely (0.022–0.048) but were comparable to those observed in the family Accipitridae (0.015–0.040). For the first time, pseudogenization of the OCA2 gene was detected in some of the studied species, based on comparisons of exon regions of the coding gene and various copies of its pseudogenes. Two pseudogene variants were identified in O. semitorques, three in B. (N.) scandiacus, and eight in B. bubo. High levels of intragenomic polymorphism were observed for OCA2 pseudogenes, driven by numerous single mutations, insertions, and deletions of varying lengths. Deep genetic differentiation was found for certain species pairs controversially assigned to the same genus, corresponding to intergeneric levels of genetic distances for OCA2 in other avian orders. This provides additional arguments in favor of their generic independence at the genus level: O. sunia – O. semitorques (0.031), B. bubo – B. (K.) blakistoni (0.024), and B. (K.) blakistoni – B. (N.) scandiacus (0.022). The phylogenetic reconstruction of Strigiformes and other taxa based on the OCA2 gene largely aligns with findings derived from other molecular markers.

The contribution of the m.1494C>T variant of the MT-RNR1 gene associated with aminoglycoside-induced deafness (MT-RNR1, OMIM 561000) to the etiology of hearing loss (HL)is still poorly studied. In this regard, aim of the study – screening of the m.1494C>T gene MT-RNR1 among patients with HL in the Republic of Buryatia located in the Baikal Lake region of Russia and reconstructed the mitochondrial lineages in 27 patients with m.1494C>T variant from different regions of the world. From available data and based on the results of a genome-wide analysis of mtDNA of one patient with m.1494C>T detected in this study we are reconstructed the mitochondrial lineages in 27 patients with m.1494C>T variant from different regions of the world. As a result, 19 different mtDNA haplogroups were identified, which likely indicates the independent origin of the m.1494C>T variant on the different mitochondrial background. However, in patients with m.1494C>T were found the high frequency of haplogroup A* (18.5%, 5/27), which in 13 times exceeded (χ² = 45.274; p < 0.001) the mean frequency of this haplogroup (1.45%, 519/35748) in worldwide population. The over representation of haplogroup A* among patients with m.1494C>T may be due of their common ancestry. The possible influence of a founder effect on the prevalence of the MT-RNR1, the target screening of the m.1494C>T variant in previously unexplored cohorts of patients with HL is a more relevant, primarily in regions where the haplogroups A* and A2 were found – in Asia and America.

The paper considers a microevolution model of two-stage population with limitation under the influence of natural selection regulating individual fertility. Analytical and numerical investigations of the model have been conducted, and parametric regions corresponding to different types of dynamic behavior have been identified. The final genetic composition of the population is shown to be determined by the reproductive potential values of heterozygotes and homozygotes. With higher productivity of heterozygotes, the model predicts stable polymorphism; under intermediate dominance, polymorphism transitions to monomorphism or the emergence of a new mutation. Reduced heterozygote reproductive potential leads to a “bistability trap”, when the system shifts to one of the possible monomorphic states depending on the initial allele frequency. Furthermore, it has been found that within a narrow range of parameter values, reduced heterozygote fertility can result in multistability, where both the bistability trap and stable polymorphism may emerge depending on initial allele frequencies. Consequently, variations in the current population structure can alter the direction of evolution. Additionally, an increase in average fertility destabilizes the population dynamics, with the nature of the resulting fluctuations being determined by ecological limitation.

The article presents the results of studying single nucleotide polymorphisms in the ACTN3 gene (rs1144978872G>A and rs1150531051T>C) in Thoroughbred horses, as well as their associations with indicators of racing performance. The protein α-actinin, which is actively involved in the contraction of muscle fibers, in mammals is represented by several genetic variants that can affect athletic abilities. Genotyping of Thoroughbred horses, carried out in the genetics laboratory of the All-Russian Research Institute for Horse Breeding, showed the presence of polymorphism in the promoter region of the ACTN3 gene. Horses with different SNP genotypes (rs1150531051T>C) differed in their speed and distance qualities, which indicates the need for further study of the associations of ACTN3 variants with performance.