Biotechnologia Acta

7-22

Authors: Yu. P. Rud, L. P. Buchatsky

Based on nucleotide sequences of sex-specific DNA-markers of salmonid fishes the oligonucleotide primers for polymerase chain reaction were selected with purpose on rapid diagnostic of sex in rainbow trout Onchorhynchus mykiss, brown trout Salmo trutta, huchen Hucho hucho and grayling Thymallus thymallus. The specify of amplification was determined by nucleotide sequence analysis of PCR-products. All amplified fragments were referred to sex-specific locuses of Y chromosomes in males of investigated fish species. The PCR-products were in size of 880, 607, 521 and 558 for rainbow trout, brown trout, grayling and huchen respectively. Thus the sex determination in above mentioned fish species and identification of genotypic males under process of hormonal sex reversion can be provided using conventional PCR. Present method relates to rapid diagnostics because the data analysis and return of results back to fish farm take one single day.

23-28

Authors: O. V. Gudzenko, N. V. Borzova, L. D. Varbanets

The study of the features of thermal inactivation of α-L-rhamnosidase from Eupenicillium erubescens aiming to develop further strategies for improving the stability of the enzyme was the purpose of the research. Two forms of α-L-rhamnosidases were studied. They were obtained by producer cultivation on two different carbon sources (rhamnose and naringin). It was shown that at temperatures above 60 0C heat inactivation process is described by the first order reaction kinetics. It was established that stability of α-L-rhamnosidase from E. erubescen, grown on naringin depends on hydrophobic interactions rather than electrostatic ones. It was shown that aggregation of enzyme molecules could lead to their thermal stabilization. The important role of cysteine residues and possible involvement of the metal cation in maintaining of active form of α-L-rhamnosidases from E. erubescens was highlighted.

40-45

Authors: G. S. Andriiash, G. M. Zabolotna, V. S. Bondarenko, S. M. Shulga

The phylogenetic relationships of strainsproducers of essential amino acids of aspartate family Brevibacterium sp. UCM Ac-674 (Brevibacterium sp. 90), Brevibacterium sp. IMV Ac-5004 (Brevibacterium sp. 90H), Brevibacterium sp. UCM Ac-675 (Brevibacterium sp. E531), mutant strain Brevibacterium sp. IMV B-7447 from the «Collections strains and lines of plants for food and agricultural biotechnology SO “Institute for Food Biotechnology and Genomics” of National Academy of Sciences of Ukraine were investigated. The affiliation strain Brevibacterium sp. IMV B-7447 to the genus Brevibacterium within the sequences of the genes based on 16S rRNA was confirmed. The dendogram of phylogenetic relationships of studied strains and related strains Brevibacterium from database GenBank was constructed. It was shown that by the criterion of homology gene sequences based on 16S rRNA the investigated strains-producers belong to three phylogenetic groups. It was established that the mutant strain Brevibacterium sp. ІMV B-7447 has no analogues in the database GenBank.

51-56

Authors: Kyryk V. M.

To develop a protocol of multiparametric phenotyping and sorting of LSK-subpopulations of hematopoietic stem cells and to determine their relative numbers in the bone marrow of mice was the goal of this research. The modified protocol of multiparametric phenotyping of murine hematopoietic stem cells enable to determine the content of Lin–Sca-1+ c-kit+, Lin–Sca-1+c-kit+flt3+CD150–, Lin–Sca-1+c-kit+flt3+CD150+ and Lin–Sca-1+c-kit+flt3–CD150– subpopulations in bone marrow of FVB mice. It was shown that the dominant population among LSK-cells represents the phenotype Lin–Sca-1+c-kit+flt3–CD150– (57.2 ± 6.8%), which characterizes the short-term hematopoietic stem cells responsible for myelopoiesis. Also the protocol of sorting of murine bone marrow LSK-cells was proposed and its effectiveness for subsequent transplantation in experiments was demonstrated. At repeated phenotyping of sorted cells the purity of Lin–Sca-1+c-kit+ cell fraction was 96.6 ± 1.8% with viability up to 89.6 ± 4.6%.

60-74

Authors: B. P. Matselyukh, L. V. Polishchuk, V. V. Lukyanchuk, S. A. Golembiovska, V. Y. Lavrenchuk

The aim of research was a comparative analysis of the sequences of the carotenoid biosynthetic gene clusters of the initial inactive strain S. globisporus 1912-2 and spontaneously arising, carotenoid producing mutant 1912-4Crt, and comparison of these sequences with the known sequences of the crt genes of the other representatives of streptomycetes. Streptomyces globisporus 1912 is a producer of the antitumor antibiotic landomycin E and new regulator of diketopiperazine nature. Comparative analysis of the obtained DNA sequences using the GenBank data allowed localization of 7 carotenoid biosynthetic crt genes of S. globisporus 1912 in one cluster. This cluster, similar to other crt clusters of different Streptomyces species, consists of two convergent operons from 4 and 3 crt genes. The high homology (93%) of the crt gene clusters of S. globisporus 1912 and S. griseus IFO 13350 was shown. Two non-punctual repeats of 21 b. p. were found in the sequence of crtY gene coding lycopene cyclase. It was shown that the deletion of 117 b.p. including the sequence between non-punctual repeats of 96 b. p. and one NPR from 5′-side activated the crt gene cluster and production of beta-carotene (6.91 mg/l) and lycopene (3.24 mg/l) by the mutant 1912-4Crt. A hypothesis about the site-specific recombination between two non-punctual repeats as the cause of the deletion in crtY gene of strain 1912-4Crt was proposed. The obtained strain 1912-4Crt is essential for the next genetic selection of the more effective carotenoid producers. Deletion of 86 b. p. was revealed in the regulatory gene lndRR resulting in the deficiency of landomycin E production by the strain 1912-4Crt. The DNA sequences of crt and lnd genes of S. globisporus 1912 were submitted to the NCBI database with accession numbers KM349312 and KJ645792, respectively.

75-82

Authors: А. I. Stepanenko, B. V. Morgun, O. V. Stepanenko, S. S. Polishchuk, O. I. Rybalka

The biotechnological analysis of modern barley varieties of national and foreign origin for the presence of valuable SE-ve genotypes was done. Identification of the allelic status of the gene was performed among 109 barley samples using the developed multiplex PCR system for SNAP molecular markers. Among the studied varieties SE+ve allele was identified in 75 barley samples and the other contained SE-ve allele. 6 samples which revealed both alleles SE+ve and SE-ve were heterogeneous (+/-). The obtained results were compared with the data of the State register of plant varieties suitable for dissemination in Ukraine and showed that 8 samples referred to as forage, contained SE+ve allele and none of them were SE-ve allele. Of 38 varieties, which quality was identified as suitable for brewing, 19 contained SE+ve allele, the rest — allele SE-ve. The results of performed analysis of the spring barley collection for allelic composition of HvITR1 gene are of great practical importance as for the correct selection of mating pairs for malting breeding, for the selection of elite plants in breeding populations, or evaluation of barley seeds for beverage brewing purposes.

92-101

Authors: S. S. Murodova, K. D. Davranov

Analysis of literature data and authors researchers on application of complex microbial preparations in agricultural practice of Uzbekistan for the purpose of soils fertility increase was the aim of the review. Application of biotechnologies on the basis of highly effective microorganisms allows offering the alternative strategy of ecologically steady land usage based on chemicals substitution by microbial preparations. Synthesis of preparations — microbial complexes from local bacterial strains, with are capable to keep the basic properties under extreme conditions is perspective direction that represents theoretical and practical interest.