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Aim. The study was aimed at cloning and analysis of molecular organization of 5S rDNA intergenic spacer (IGS) in two Gentiana species of Ukrainian flora, G. pneumonanthe L. and G. punctata L. Methods. 5S rDNA IGS sequence was amplified using polymerase chain reaction (PCR) with a pair of primers specific for the gene coding region. The produced PCR products were fractionated by gel-electrophoresis, isolated, ligated into plasmid pUC18, cloned into E. coli, and then sequenced. Nucleotide sequences were aligned using the Muscle algorithm and analyzed in the Unipro UGENE software. Results. The intergenic spacer region of the 5S rRNA genes was cloned and sequenced for two Gentiana species of Ukrainian flora, G. pneumonanthe and G. punctata. Based on the analysis of the alignment of the IGS sequences of five Gentiana species from three sections, some features of molecular organization of IGS of 5S rRNA genes in the studied species were established. In particular, motifs typical for other angiosperm families were identified, such as conservative oligo-dT motif at the IGS 3'-end that served as a transcription termination site and AT-rich region preceding the coding region of 5S rRNA gene. However, in the region of transcription initiation, conservative GC-element in position -13 is changed to AC. Conclusions. The interspecific variation of molecular organization of 5S rDNA IGS was identified among Gentiana species that can be used to clarify the phylogenetic relationships between members of this genus.Keywords: Gentiana species, 5S rDNA intergenic spacer, molecular organization, phylogeny.

Retrotransposons make up a significant part of plant genome and are probably the most dynamic part of it, so they play a significant role in the generation of genetic variation. In particular, their activation can lead to structural reorganization of genome and changes in genome size, the emergence of novel genetic and phenotypic variants, as well as changes in gene expression, thus providing the raw material for adaptation and evolution. This review summarizes literature data on the activation of LTR-retrotransposons of the superfamilies Ty1/Copia and Ty3/Gypsy during in vitro culture and under various abiotic and biotic stress conditions. Their structure, classification, and significance for the organization and functioning of plant genome are reviewed. The main mechanisms of activation of LTR-retrotransposons under stress conditions are explored, including changes in DNA methylation and interaction of stress-induced transcription factors with retrotransposon promoters due to the presence of specific binding sites and other regulatory elements. The review also discusses consequences of activation of retrotransposons and control of their activity by self-inactivation mechanisms and the epigenetic regulation of genome.Keywords: retrotransposons, Ty1/Copia, Ty3/Gypsy, in vitro culture, abiotic and biotic stress.

Aim. Peroxisome proliferator activated receptor gamma (PPARG) and PPARG coactivator 1α (PPARGC1A) is a key regulator of energy metabolism. This study examines the influence of PPARG and PPARGC1A gene polymorphisms on the PPARG expression, obesity risk, lipoprotein profile and effectiveness of the physical activity intervention for improvement of these parameters. Methods. 39 women with BMI>30 kg/m2 participated in the three-months fitness-program and followed a hypocaloric diet (1500 kCal). At the beginning and at the end of the program, the following anthropometric and biochemical parameters were measured: BMI, percentage of total and visceral fat, amount of plasma lipoproteins, cholesterol, and triglycerides. Single nucleotide polymorphisms were identified in PPARG (n=94) and PPARGC1A (n=138) genes. PPARG mRNA expression was measured through reverse transcription PCR. Results. The physical exercise intervention resulted in a significant fat mass loss in all participants (40.3±5.3% before the study vs 36.4±5.7% after the study, P

Aim. The aim of the study was a comparative assessment of total phenolic content and total flavonoid content in in vitro plants, regenerated plants, plants grown in a growth chamber, and tissue culture of several genotypes of Deschampsia antarctica. Methods. In vitro culture, Folin-Ciocalteu method, spectrophotometry, high-performance liquid chromatography. Results. The total content of phenolic compounds and total flavonoid content was determined in the samples of three D. antarctica genotypes: G/D12-2a (2n=26), DAR12 (2n=26+0–3B) and Y66 (2n=36–39). The content of these biologically active compounds was the highest in in vitro plants and it was higher than in plants grown in a growth chamber. The highest content of BAC was found in DAR12 in vitro plants (16.50 mg of ferulic acid equivalent and 21.26 mg of rutin equivalent per g of dry weight, respectively). The regenerated plants did not differ significantly in the content of BAC from the original in vitro plants. In tissue culture, the content of BAC was lesser. One- and two-year-old tissue cultures did not differ significantly in the content of phenolic compounds and flavonoids. Conclusions. The relatively high content of phenolic compounds and flavonoids in in vitro plants and in regenerated plants indicates that in vitro cultivated D. antarctica plants can be a promising raw material for production of valuable BACs. Keywords: Deschampsia antarctica E. Desv., in vitro plants, plant tissue culture, phenolic compounds, flavonoids.

Aim. Identification of Ty1/Copia-like transposable elements in Deschampsia antarctica E Desv. in silico. Methods. Bioinformatic analysis of sequence read archives of D. antarctica genome and transcriptome contained in the GenBank database was conducted. The search was carried out using the rice Ty1/Copia TE Tos 17 as a reference sequence. Results. The search revealed the sequences of Ty1/Copia retrotransposons in the D. antarctica genome to show a high level of identity (up to 75% in the homologous regions) to Tos 17. The uneven distribution of the found reads along the reference sequence indicates the existence of a group of the sequences in the genome to contain elements typical of the family and have varying degrees of identity to Tos 17, with more conservative ones being represented in correspondingly greater numbers among the found reads. The presence of the reads identical to Tos 17 in transcriptome indicates that the TE sequences identified in genome have a certain background level of transcriptional activity. Conclusions. Transcriptionally active Ty1/Copia-like transposable elements were identified in silico in D. antarctica genome using methods of bioinformatics. The sequences found can be used to construct primers for PCR and to analyze the activity of the TE of this family in D. antarctica in further studies. Keywords: bioinformatic analysis, Deschampsia antarctica E Desv., transposable elements, Ty1/Copia.

Aim. The study was aimed at cytogenetic analysis of Iris pumila, I. attica, and I. pseudopumila, comparative study of the karyotypes of these species, as well as identification of putative origin of I. pumila karyotype. Methods. Cytogenetic analysis of root apical meristem, determination of chromosome number in mitotic metaphase plates, anaphase analysis. Results. The chromosome numbers observed were 2n=32 for I. pumila plants from different localities in Ukraine and 2n=16 for I. attica and I. pseudopumila plants from Greece and Italy, respectively. Some of the plants were mixoploids, the smallest proportion of mixoploids was in I. pseudopumila (10.9%) and the largest in I.pumila from all studied populations (60-80%). Anaphase analysis showed the presence of chromosomal aberrations in 2.6% of cells in roots of I. pseudopumila seedlings. The highest level of structural chromosomal aberrations (9.2%) was found in root apical meristem cells of I. pumila seedlings. Conclusions. The chromosome number was established as 2n=32 for I.pumila plants and 2n=16 for I. attica and I. pseudopumila plants. The high level of mixoploidy (60–80% of mixoploid plants) and anaphase chromosomal aberrations (up to 9.2%) was found in apical meristem of I. pumila seedlings. The amphidipiloid nature of I. pumila was established; the karyotype of the species could be formed as a result of a combination of chromosome sets from hypothetical ancestral species I. attica and I. pseudopumila. Keywords: Iris pumila L., Iris attica Boiss. & Heldr., Iris pseudopumila Tineo, chromosome number, amphidiploid, mixoploidy.

Aim. The work was aimed at the development of conditions for introduction into in vitro culture of two species of irises, Iris attica and I. pseudopumila to obtain aseptic seedlings with subsequent reintroduction into natural environment, as well as at cytogenetic analysis of the obtained plants. Methods. In vitro seed germination and seedling cultivation. Cytogenetic analysis of cells of root meristem, determination of chromosome number and morphology in mitotic metaphase plates, anaphase analysis. Results. The plants of I. attica and I. pseudopumila were introduced in vitro. Aseptic seedlings were obtained, which were actively growing on MS/2 medium without phytohormones. The experiments on the adaptation of the plants to greenhouse conditions revealed the high survival rate for both species. The chromosome number 2n = 16 was established for the obtained plants of both I. attica and I. pseudopumila. Mixoploidy was detected in root meristem of some of the plants, the incidence of which was 10.9 % for I. pseudopumila and 30.4 % for I. attica. The frequency of cells with chromosomal rearrangements revealed by anaphase analysis in root meristem of I. pseudopumila seedlings was 2.6 %; in I. attica plants, chromosome aberrations were not detected. Conclusions. The plants of I. attica and I. pseudopumila were introduced into in vitro culture, aseptic seedlings were obtained, which showed a high survival level when adapted to greenhouse conditions. Chromosome number 2n = 16 was established for the obtained plants of both species. The root apical meristems of the seedlings were found to be mixoploid, with the incidence of mixoploidy in I. attica identified as three times higher than in I. pseudopumila plants.Keywords: I. attica Boiss. & Heldr., I. pseudopumila Tineo, aseptic seedlings, mixoploidy, anaphase aberration.

The origin of one from two native vascular plants in Antarctic, the Antarctic hairgrass (Deschampsia antarctic E. Desv.) (Poaceae), remains unclear. Molecular genetic analyses have demonstrated low genetic heterogeneity among representatives of this species. The search for new marker nucleotide sequences of the Antarctic hairgrass is justified, which would be useful in clarifying the phylogeographic origin, heterogeneity and population dynamics of D. antarctica. Here we report the results of a comparative analysis of the introns of β-tubulin genes for several populations of the Antarctic hairgrass across the maritime Antarctic. We demonstrate that the first intron of the β-tubulin gene contain markers capable of discriminating D. antarctica subpopulations in the maritime Antarctic. The sequence of the first intron of the β-tubulin gene can therefore be used for more detailed population genetics studies of D. antarctica.

Obesity is a widespread problem within modern society, serving to increase the risk of cardiovascular, metabolic, and neurodegenerative disorders. Peroxisome proliferator-activated receptor gamma (PPARγ) and PPARγ coactivator 1 α (PGC1α) play a key role in the regulation of cellular energy metabolism and is implicated in the pathology of these diseases. This study examined the association between polymorphisms of the PPARG and PPARGC1A genes and individual variability in weight loss in response to physical activity intervention. 39 obese Ukrainian women (44.4 ± 7.5 years, BMI > 30.0 kg/m2) undertook a 3-month fitness program whilst following a hypocaloric diet (~ 1500 cal). Anthropometric and biochemical measurements took place before and after the program. Single nucleotide polymorphisms within or near PPARG (n = 94) and PPARGC1A (n = 138) were identified and expression of PPARG mRNA was measured via reverse transcription and amplification. The association between DNA polymorphisms and exercise-induced weight loss, initial body mass, biochemistry and PPARG expression was determined using one-way analysis of variance (ANOVA). The present intervention induced significant fat loss in all participants (total fat: 40.3 ± 5.3 vs 36.4 ± 5.7%; P

D. antarctica E. Desv. plants cultivated in vitro were analyzed for the chromosome number, leaf length, and efficiency of callus formation. Most of the studied plants had a typical diploid chromosome number. However, a hypotriploid and a plant with B chromosomes demonstrated mixoploidy caused by the presence of a significant proportion (up to 15–25%) of aneuploid cells. Jaccard genetic distances between the plants determined from the data of ISSR- and IRAP-PCR analyses were within the range of 0.0323 to 0.1803. Furthermore, genetic distances between the specimens with atypical karyotype and diploids did not exceed the paired distances within the group of diploid plants. Variations in the leaf length and growth parameters of the plants were characterized. Plants with different chromosome numbers differed in the leaf length and efficiency of callus formation. Obtained results may indicate relationships between the chromosome number, studied morphometric parameters, and efficiency of callus formation in the analyzed D. antarctica plants.

A comprehensive study of a typical steppe perennial Iris pumila L. were carried out in the central zone of the European part of this species’ range, namely in Ukraine. Intraspecific differentiation, population size and isolation degree and its consequences, the threat of human impact were analyzed, as well as ecological amplitude and genetic variation in ISSR markers and selected chloroplast regions were determined. The species was found to have a low intraspecific differentiation that indicates the uniformity of the gene pool in the studied part of the range. Moreover, the results of isolation assessment, population and ecological study of I. pumila confirm the potential risk of extinction. A considerable part of the species populations exist as separated patches of rare ecosystems, isolated from the nearest neighbours due to intense plowing of the steppe zone. The generative reproduction is rare. In contrast, ISSR analysis revealed comparably high genetic diversity in all the sampled populations. Furthermore, specific plastid haplotypes were demonstrated in some of them. The inconsistency between the results of population ecological study and the data of molecular genetic analysis indicates that the loss of genetic diversity in the species caused by habitat fragmentation and isolation under increasing anthropogenic pressure is likely to be slower than it would appear judging from the assessment of population parameters, which clearly show negative trends. This result also emphasizes the necessity for integrated approach to assessment of the extinction risk for particular species and careful analysis of all determinants of extinction.

Aim. To clarify the details of chromosome variation in calli derived from D. antarctica plants in the initial passages of the culture in vitro. Methods. Induction of callus from root explants of plants, which were grown from seeds, and consequent subcultivation of tissue culture. Cytogenetic analysis of squashed slides stained by acetic-orcein and counting the number of chromosomes in mitotic metaphase plates. Results. There were analyzed the cultured tissues derived from D. antarctica plants with different chromosome numbers: diploid plants (2n=26), mixoploid plant with B-chromosomes (2n=26+1-3B), and mixoploid plant with near-triploid modal class (2n=36, 38). Analysis of callus tissues of all plants at 2-4 passages revealed mixoploidy, presence of polyploid and aneuploid cells. The modal class in all studied calli was composed of diploid and aneuploid cells with near-diploid chromosome number. The cytogenetic structure of cell population of cultured tissues was found to vary with characteristics of the karyotype of donor plant. The largest range of variation in the number of chromosomes (from 18 to 63 chromosomes) was found in tissue culture of diploid plant (2n=26) from the Galindez Island, and the highest frequencies of polyploid (47 %) and aneuploid cells were in the culture of mixoploid plant with near-triploid modal class from the Big Yalour Island. Conclusions. In different D. antarctica cultured tissues at the early stages of the culture, the modal class was composed of diploid cells and cells with near-diploid chromosome number irrespective of karyotype of donor plant (diploid, mixoploid poliploid).Key words: Deschampsia antarctica Desv., plant tissue culture, chromosomal variability in vitro, mixoploidy.

Aim. To study the potential effects of different concentrations of cadmium ions on antarctic plant D. antarctica using PCR analysis. Methods. Plants were grown in vitro on B5 Gamborg and Eveleigh agar medium supplemented with CdCl2. Genetic rearrangements were studied by PCR-analysis using ISSR- and IRAP-primers. Results. Genetically identical plants of D. antarctica obtained by microclonal propagation in vitro were used for the study of mutagenic effect of cadmium ions. The influence of Cd2+ was investigated within the concentration ranging from 0.1 to 10 mM. The results of cultivation of D. antarctica plants in the presence of cadmium ions for 63 days allow to determine the concentration range that does not inhibit the growth of the plants in vitro, and it was up to 1 mM. It was found that toxicant concentrations of 0.1 and 0.2 mM did not cause changes in the profiles of PCR products. After growing the plants with 0.2–1 mM CdCl2 for 17 days, the changes in the profiles of PCR products, indicating the mutagenic impact, were observed at concentrations of 0.6 mM or above; moreover, the number of changes increased in dependence on the concentration of heavy metal. Prolonged influence (140-265 days) of cadmium ions in relatively low concentrations (0.1 mM and 0.4 mM) did not cause detectable mutations. Conclusions. D. antarctica, a plant extremophile, which has evolved mechanisms of resistance to a variety of extreme conditions as a result of adaptation to the existence in the harsh conditions of Antarctica, shows enhanced resistance to cadmium ions in comparison with other species of vascular plants. Inhibition of growth occurs at Cd2+ concentrations of 0.1 mM or above, whereas concentrations of 1 mM or above cause cessation of growth and death of plants. Mutagenic effect on D. antarctica was observed at Cd2+ concentrations of above 0.4 mM. After prolonged growth of plants (for 3–8 months) at cadmium ions concentrations of 0.1–0.4 mM, genetic changes was not found.Keywords: Deschampsia antarctica Desv., plants in vitro obtained by microclonal propagation, cadmium ions, mutagenic effect, PCR-analysis.

Cytogenetic analysis of Iris pumila L. plants from different habitats in Ukraine revealed the intrachromosomal polymorphism. Chromosome number 2n = 32 was established for plants of this species. Root meristem of I. pumila seedlings of all populations showed high level of mixoploidy. The differences in the percentage of aneuploid cells and anaphase aberration were found between the sampled populations. Anaphase analysis revealed the level of chromosomal rearrangements that reached 9.2% in some populations reached.

The aim of the study was to develop the system of polymerase chain reaction (PCR)-based markers for the assessment of genetic diversity and population genetic studies of Gentiana lutea L. as well as to determine the utility of two indices of marker informativeness. The informativeness was determined for 40 PCR primers of different types (random amplified polymorphic DNA, inter simple sequence repeat, inter-retrotransposon amplified polymorphism, resistance gene analog polymorphism and conserved DNA-derived polymorphism markers) by evaluating discriminating power (DL) and resolving power (Rp) in a sample of 30 plants from two populations. Analysis of correlation between the index value and the number of differentiated pairs of genotypes in the given sample revealed that DL is more efficient than Rp; therefore, we selected primers based on the DL value. In total, 12 primers with the largest values of DL were chosen. Analysis of genetic relationship among 86 plants from six populations showed that the number of bands produced by the three of selected primers was sufficient to give average bootstrap support across six key nodes in the dendrogram higher than 85%, while using six of the primers resulted in average bootstrap value exceeding 99%. Thus, a minimal set of three to six selected primers are sufficient for a quick assessment of genetic diversity of G. lutea populations, depending on the sample size and degree of differentiation between populations, while the rest of the primers with DL values above 0.8 may be used for ecogenetic surveys. Preliminary results obtained with selected primers indicate the moderate level of genetic variation within the species and significant differentiation among individual populations.

We investigated informativeness and effectiveness of different marker types (ISSR, IRAP, REMAP, RGAP and LP-PCR that employ primers based on the conservative sequences of abiotic stress response genes) to study genetic diversity of Iris pumila L. By the number of amplicons per primer, number of polymorphic amplicons per primer and resolving power index (Rp), ISSR-markers were the most efficient followed by LP-PCR-markers. In order of decreasing value of indicators of genetic diversity “the percentage of polymorphic bands”, and “the average Jaccardś genetic distance between plants”, marker systems may be arranged as follows: ISSR > RAPD > LP-PC > RGAP ≈ IRAP. For ISSR-markers, the percentage of polymorphic bands was 1.3–1.7 times higher than for the others, and the average genetic distance was 1.2–1.3 times higher. Different marker systems were ranked by the value of Neiś gene diversity and the Shannonś index as follows: ISSR > RAPD ≈ LP-PCR > RGAP ≈ IRAP, with the highest and the lowest values differing 1.4 times. Genetic population structure was investigated with program Structure 2.3. The data of all marker systems suggest that all genomes under study belonged to one population. The PCoA and cluster analyses based on genetic distances showed distinctions in clustering generated from different markers data and summarized data, as well as the lack of strong clusters. Mantel test revealed significant positive correlation between the matrices of genetic distances generated by the data of almost all marker systems. The strongest correlation was found between RGAP- and IRAP-markers (r = 0.452, p = 0.01) and between RGAP and ISSR (r = 0.430, p = 0.01). ISSR, RAPD and LP-PCR proved to be more effective for the study of I. pumila genetic diversity, nevertheless, joint use of different marker systems will provide a more comprehensive assessment of variation in different genomic regions.

The genus Deschampsia P. Beauv (Poaceae) involves a group of widespread polymorphic species. Some of them are highly tolerant to stressful and variable environmental conditions, and D. antarctica is one of the only two vascular plants growing in Antarctic. This species is a source of useful for selection traits and a valuable model for studying an environmental stress tolerance in plants. Genome diversity and comparative chromosomal phylogeny within the genus have not been studied yet as karyotypes of most Deschampsia species are poorly investigated. We firstly conducted a comparative molecular cytogenetic analysis of D. antarctica (Antarctic Peninsula) and related species from various localities (D. cespitosa, D. danthonioides, D. elongata, D. flexuosa (= Avenella flexuosa), D. parvula and D. sukatschewii by fluorescence in situ hybridization with 45S and 5S rDNA, DAPI-banding and sequential rapid in situ hybridization with genomic DNA of D. antarctica, D. cespitosa, and D. flexuosa. Based on patterns of distribution of the examined markers, chromosomes of the studied species were identified. Within these species, common features as well as species peculiarities in their karyotypic structure and chromosomal distribution of molecular cytogenetic markers were characterized. Different chromosomal rearrangements were detected in D. antarctica, D. flexuosa, D. elongata and D. sukatschewii. In karyotypes of D. antarctica, D. cespitosa, D. elongata and D. sukatschewii, 0-3 B chromosomes possessed distinct DAPI-bands were observed. Our findings suggest that the genome evolution of the genus Deschampsia involved polyploidy and also different chromosomal rearrangements. The obtained results will help clarify the relationships within the genus Deschampsia, and can be a basis for the further genetic and biotechnological studies as well as for selection of plants tolerant to extreme habitats.

In order to study the genetic diversity of D. antarctіca Desv. and phylogenetic relationships between the plants from Antarctica and outside of its borders RAPD-analysis and comparative sequence analysis of rDNA internal transcribed spacers 1 and 2 region (ITS1-2) were conducted. There are 15 samples were studied from two distant regions, namely from King George Island and from islands of Argentine archipelago region. The results of RAPD-analysis indicate the differentiation of the studied populations at the molecular genetic level and the existence of a geographical gradient of genetic diversity of D. antarctіca in Maritime Antarctica with a decrease in this parameter from north to south. Sequence analysis of the ITS1-2 rDNA region revealed the presence of several variants in Antarctic populations that differ by specific mutations from plants growing outside Antarctica.

It is established that switching from surface to submerged cultivation in a liquid medium of a special composition does not induce noticeable changes in the callus tissues of Rauwolfia serpentina in the course of the first several passages. Polymorphism of the RAPD spectra is discovered following 4–6 growth passages of tissues in the submerged culture; this polymorphism may reflect both changes in the DNA sequence and in the genetic structure of the cell population forming the strain. Addition of an intermediate passage in a solid medium of simpler composition prior to a switch to the liquid medium does not exert a substantial influence on the level and nature of the changes in the genome.

18S-25S rDNA of intact plants and tissue cultures of G. acaulis, G. punctata, and G. lutea are studied by blot hybridization. A decrease in the number of repeats of ribosomal DNA by comparison with the plants is established in the callus tissues. Unlike other species, G. lutea exhibits intragenome heterogeneity of rRNA genes as well as qualitative changes in rDNA in tissue cultures, in particular, the appearance of altered repeats. It is suggested that there exists an association between these features of the structure of rRNA genes and their rearrangement in vitro.

Cytogenetic analysis of Iris pumila L. plants from different habitats in Ukraine revealed the intrachromosomal polymorphism. Chromosome number 2n = 32 was established for plants of this species. Root meristem of I. pumila seedlings of all populations showed high level of mixoploidy. The differences in the percentage of aneuploid cells and anaphase aberration were found between the sampled populations. Anaphase analysis revealed the level of chromosomal rearrangements that reached 9.2% in some populations reached.

Deschampsia antarctica Desv. (Poaceae) (2n = 26) is one of the two vascular plants adapted to the harshest environment of the Antarctic. Although the species is a valuable model for study of environmental stress tolerance in plants, its karyotype is still poorly investigated. We firstly conducted a comprehensive molecular cytogenetic analysis of D. antarctica collected on four islands of the Maritime Antarctic. D. antarctica karyotypes were studied by Giemsa C- and DAPI/C-banding, Ag-NOR staining, multicolour fluorescence in situ hybridization with repeated DNA probes (pTa71, pTa794, telomere repeats, pSc119.2, pAs1) and the GAA simple sequence repeat probe. We also performed sequential rapid in situ hybridization with genomic DNA of D. caespitosa. Two chromosome pairs bearing transcriptionally active 45S rDNA loci and five pairs with 5S rDNA sites were detected. A weak intercalary site of telomere repeats was revealed on the largest chromosome in addition to telomere hybridization signals at terminal positions. This fact confirms indirectly the hypothesis that chromosome fusion might have been the cause of the unusual for cereals chromosome number in this species. Based on patterns of distribution of the examined molecular cytogenetic markers, all chromosomes in karyotypes were identified, and chromosome idiograms of D. antarctica were constructed. B chromosomes were found in most karyotypes of plants from Darboux Island. A mixoploid plant with mainly triploid cells bearing a Robertsonian rearrangement was detected among typical diploid specimens from Great Jalour Island. The karyotype variability found in D. antarctica is probably an expression of genome instability induced by environmental stress factors. The differences in C-banding patterns and in chromosome distribution of rDNA loci as well as homologous highly repeated DNA sequences detected between genomes of D. antarctica and its related species D. caespitosa indicate that genome reorganization involving coding and noncoding repeated DNA sequences had occurred during the divergence of these species.

An analysis of 18S-25S and 5S rRNA genes in intact plants and cultured tissues of some Rauwolfia species was performed to compare these sequences variability occurred as a result of the species evolution in nature and that induced by tissue culture. The restriction fragment length polymorphism of 18S-25S and 5S rDNA was found both in intact plants of various Rauwolfia species and in long-term Rauwolfia serpentina tissue cultures. In addition, changes in the amount of 18S-25S rRNA genes were observed in long-term R. serpentina tissue cultures. The results demonstrate that rDNA variability observed in intact plants as well as in long-term cultures is attributed to differences in the same regions of ribosomal RNA genes.

To investigate the processes that occur in the embryo cell nuclei in the course of natural and accelerated aging of rye seeds, nuclear DNA structural organization into chromatin loop domains was studied. The loss of germination was shown to be accompanied by a decreased excision of chromatin loop domains. The study of chromatin accessibility to DNase I did not reveal any considerable changes in chromatin architecture that would explain the decreased DNA fragmentation at matrix attachment regions. A soluble nuclear protein of ca. 31 kD was found to manifest nuclease activity, which declined with the loss of germination. The study of DNA fragmentation in histone-depleted nuclei (nucleoids) disclosed a nuclease activity resistant to 2 M NaCl extraction and sensitive to the specific inhibitors of DNA topoisomerase II; the latter activity also declined with aging. The authors conclude that the changes in DNA fragmentation patterns in aging seeds were primarily caused by a decreased activity of the enzymes accounting for the excision of chromatin loop domains.

This chapter presents the results of cytogenetic and molecular genetic studies on tissue and organ cultures from some Gentiana species. Cytogenetic analysis of calli from seven gentians showed that all of them (except for G. punctata ) consisted mainly of the cells with diploid and/or near-diploid chromosome sets with low anaphase aberration. Species specificity of cytogenetic structure was established for cell populations in vitro. An increase in the proportion of polyploid cells was found for G. acaulis callus after long-term culture. RAPD analysis of tissue cultures from six gentians showed that culture resulted in genetic changes, with the species differing in the level of genetic variation. RAPD and ISSR analyses of G. pneumonanthe and G. lutea genetic variation in tissue and organ cultures and regenerated plants revealed the most significant changes in isolated root cultures as well as in long-term cultured tissues. At the same time, direct and indirect regenerants had the least genetic difference compared to donor plant . The restriction maps of nuclear 18S-25S ribosomal DNA from G. acaulis , G. punctata , G. lutea, and G. asclepiadea were constructed based on the results of blot hybridization. The decrease of 18S-25S rDNA amount was found in cultured tissues. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, such as the appearance of repeats with altered length. A relationship has been suggested between the peculiarities of the structural organization of the 18S-25S rRNA gene cluster and its rearrangements. Rearrangements of 5S rDNA were not found in cultures of Gentiana L. species, except for the variation in nucleotide methylation pattern at restriction sites.

Comparative analysis of the sequence of the ITS1-2 rDNA region of Deschampsia antarctica showed that plants of this species with different genotypes may have migrated into Antarctica after the glacial period. The use of RAPD markers allowed the identification of differences at the level of polymorphisms between populations of D. antarctica located in different latitudes and showed a limitation of exchange of genetic material between these populations.

Background The production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture. Results Since HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro. Conclusions Engineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine.

The treatment of agarose embedded nuclear or cellular preparations with protein denaturing agents resulted in ordered cleavage of intact nuclear DNA into high molecular weight fragments with the pattern of fragmentation being unityped for various eukaryotic representatives. We snowed that the set of DNA fragments represents the pre-existing DNA structural domains attributed to the higher levels of chromatin folding, and presented evidence allowing to interpret the nuclear DNA domain organization as a constituent component of topoisomerase III DNA complex with its ability to mediate the cleavage/religation reactions. We demonstrated that changes in the integrity of nuclear DNA, recognizable as an altered pattern of SDS-dependent cleavage of nuclear DNA into high molecular weight DNA fragments, took place at the early stage of apoptosis, upon number of stress challenges and in cells showing various proliferative status. The changes in the integrity of nuclear DNA affected by various influences were shown to be prompt and seem to be of transient nature. The results obtained allow to conclude that changes in the integrity of nuclear DNA revealed as an altered pattern of SDS-dependent high molecular weight DNA cleavage may present the specific genome reaction accompanying the physiological changes in the cells during apoptosis, stress response and differentiation. Уданій роботі показано, що при дії на «заплавлені» в агарозу препарати клітин та ядер білкових денатурантів відбувається упорядковане крупноблочне розщеплення інтактної ядерної ДНК. Фрагменти, що утворюються при цьому, являють собою передіснуючі структурні домени ядерної ДНК, які відповідають виищм рівням упакування хроматину. їх можна розглядати як конститутивний компонент ядерного комплексу ДНК/топоізомераза II, спроможного здійснювати реакцію розщеплення – воз'єднання ДНК Встановлено, що зміна нативності, або цілісності, ядерної ДНК, яка виявляється у зміні характеру DS-Na-золежного крупноблочного розщеплення, ядерної ДНК, відбувається на ранніх етапах апоптозу під дією різноманітних стресових факторів, а також у клітинах з різним рівнем проліферативної активності. Отримані результати дозволяють припустити, що спостережені зміни нативності ядерної ДНК можуть бути специфічною геномною реакцією, яка супроводжує фізіологічні процеси в клітині приаіюптозі, відповіді на стрес або діференціювання. В данной работе показано, что воздействие на препараты «заплавленных» в агарозу клеток или ядер белковых денатурантов приводит к упорядоченному крупноблочному расщеплению интактной ядерной ДНК. Фрагменты, образующиеся при этом, являют собой предсуществующие структурные домены ядерной ДНК, соответствующие высшим уровням упаковки хроматина. Их можно рассматривать как конститутивный компонент ядерного комплекса ДНК/ топоизомераза II, способного осуществлять реакцию расщепления/воссоединения ДНK Установлено, что изменение нативности, или целостности, ядерной ДНК, проявляющееся в изменении характера DS-Na-зависимого крупноблочного расщепления ядерной ДНК, происходит на ранних этапах апоптоза под влиянием всевозможных стрессовых факторов, а также в клетках с различным уровнем пролиферативной активности. Эти изменения происходят быстро и могут иметь обратимый характер. Полученные результаты позволяют предположить, что наблюдаемые изменения нативности ядерной ДНК могут представлять собой специфическую геномную реакцию, сопровождающую физиологические процессы в клетке при апоптозе, ответе на стресс или дифференцировке.

We studied the peculiarities of nuclear DNA fragmentation in primary culture of marine thymocytes and in human lymphoblastoid cell culture (line CEM) induced to apoptosis by various influences. We showed that in apoptotic cells the ordered high molecular weight DNA cleavage, recognizable as DNA fragment release of 50–100 and 250–300 kb in length, preceded to typical nuclear DNA fragmentation at the internucleosomal regions. Comparative study of the high molecular weight and internucleosomal DNA fragmentation demonstrated that these show different sensitivity to Zn²⁺ ions, actinomycin D and cycloheximide. In addition, the formation of large molecular weight fragments in contrast to oligonucleosomal ones may be reverted in conditions promoting to topoisomerase 11 mediated rejoining of cleaved DNA. These finding suggest that the high molecular weight and internucleosomal fragmentation may follow two different program¬mes of DNA cleavage during apoplosis mediated, presumably, by diverse enzymes. We showed that similar processes of ordered high molecular weight DNA cleavage occur in apoptotic cells and in those subjected to the stress challenges. The formation of high molecular weight DNA fragments was demonstrated to proceed promptly, show no correlation with cell death and occassionally may be of transient nature. The data obtained allow to interpret the formation of high molecular weight fragments as a component of cell response to stress rather that an early step of DNA fragmentation during apoptosis. В роботі досліджено особливості фрагментації ядерної ДНК у первинній культурі тимоцитів мишей і в культурі лімфобластоїдних клітин людини (лінія СЕМ), індукованих до апоптозу різними чинниками. Крім того, наведено дані про те, що процес форму­ вання великих фрагментів за своїми характеристиками відрізняється від міжнуклеосомної фрагментації і спостерігається не лише при апоптозі, але і в клітинах, підданих дії стресу. Зміна нативності ядерної ДНК у відповідь на стрес відбувається швидко, не є пов'язаною з загибеллю клітин і в деяких випадках їй притаманний транзитний характер. Отримані результати дозволяють інтерпретувати формування великих фраг­ментів скоріше як компонент клітинної відповіді на стрес, а не як ключову властивість апоптозу. В работе исследованы особенности фрагментации ядерной ДНК в первичной культуре тимоцитов и в культуре лимфобдастных клеток человека (линия СЁМ), индуцированных к апоптозу различными воздействиями. Кроме того, приведены данные о том, что процесс формирования крупных фрагментов по своим характеристикам отличается от межнуклеосомной фрагментации и имеет место не только при апоптозе, но и в клетках, подверженных стрессовым воздействиям. Изменение нативности ядерной ДНК в ответ на стресс происходит быстро, не связано с гибелью клеток и в ряде случаев носит транзитный характер. Полученные результаты позволяют интерпретировать формирование крупных фрагментов скорее как компонент клеточного ответа на стресс, а не как ключевую характеристику апоптоза.

International audience; During the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3) synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical. Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd) shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59-61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1-47), as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo.

Genome of Rauwolfia serpentina callus cells was found to fail undergo the noticeable changes for several early passages upon the switch from surface to submerged cultivation in the liquid medium of special composition. After subsequent 4-6 passages in submerged culture RAPD spectra polymorphism was revealed which may reflect the changes in DNA sequence as well as in the structure of cell population that forms the strain. Introduction of the intermediary passage on the agar-solidified medium of more simple composition prior to transfer into liquid medium appeared not to affect essentially the level and the pattern of genome changes.

In this paper we demonstrate that apoptosis in primary culture of murine thymocytes and in continuously growing human cells is associated with the progressive disintegration of nuclear DNA into high molecular weight (HMW)–DNA fragments of about 50–150 kb. We also show that the formation of similarly sized HMW–DNA fragments takes place in the same cells in the absence of apoptotic inducers. Unlike an apoptotic fragmentation of nuclear DNA, the formation of HMW–DNA fragments in nonapoptotic cells is rapidly induced, has no correlation with the cell death, and is not associated with the development of oligonucleosomal “ladder” or apoptotic changes in nuclear morphology. The disintegration of DNA into HMW-fragments is also observed in nuclei isolated from healthy, nonapoptosizing tissues of various eukaryotes. We show that the formation of HMW–DNA fragments in the absence of apoptotic inducers is strongly dependent on the ionic detergents, is responsive to the topoisomerase II-specific poison, teniposide, and is completely reversible under conditions that favor topoisomerase II-dependent rejoining reaction. Also, we demonstrate that the formation of HMW–DNA fragments in continuously growing cell lines caused either by serum deprivation or monolayer establishment is of a transient nature and rapidly reverses to the control level following serum addition or dilution of monolayer. The results suggest that the cleavage of nuclear DNA into HMW–DNA fragments is associated not only with apoptosis but also accompanies changes in functional activity of nonapoptotic cells.

Only two vascular plants, Deschampsia antarctica Desv. (Poaceae) and Colobanthus quitensis Kunth. Bartl. (Caryophyllaceae), inhabit the Antarctic. To clarify the taxonomic position, phylogeographic origin, genetic heterogeneity, and population dynamics of D. antarctica, we comparatively analyzed the ITS1 and ITS2 sequences for several populations from two geographically distant regions of the maritime Antarctic (the South Shetland Islands and the Argentine archipelago). All accessions of D. antarctica formed a strongly supported clade in the phylogenetic dendrograms constructed. Despite the high degree of sequence similarity at ITS1eITS2 (97%e100%), the populations of D. antarctica in Tierra del Fuego, Falkland Islands and Antarctic can be discriminated at the molecular level. Our data indicate that the majority of D. antarctica populations originated from South America. Different populations may have invaded Antarctic at different times. Genetically distinct plants may coexist within the same or adjacent populations on Antarctic islands. 2010 Elsevier B.V. and NIPR. All rights reserved.

The ChK-218 line was obtained by means of experimental mutagenesis, that is, treatment of the seeds of the VIR-27 line with streptomycin. Significant differences between these lines in terms of the dimensions and number of heterochromatin regions in different pairs of chromosomes have been previously demonstrated. In the present study a comparative molecular and genetic analysis of the genomes of the VIR-27 and ChK-218 lines is carried out using SSR-and RAPD-polymerase chain reaction. The degree of polymorphism is shown to be 26.9% for the SSR alleles and 21.7% for the RAPD amplicons. These differences seem to indicate that streptomycin treatment of maize seeds produces a noticeable mutagenic effect, moreover, besides the structure of the chromosomes, changes also occur in the DNA sequence.

Nuclear DNA is arranged into loop domains by periodical attachment of chromatin fibers to nuclear matrix at highest level of chromatin compactisation. Chromatin loops represent the basic structural components of higher order chromatin folding, which are maintained during cell cycle and in differentiated cells. An increasing number of evidences suggests strong relationship between chromatin structure compactisation and functional activity of cell nucleus. We showed previously that regular DNA cleavage into high molecular weight (HMW) DNA fragments can be induced in intact nuclei, resulting in formation of ~300 kb and 50–100 kb fragments. The patterns of induced cleavage were essentially similar both in intact and high-salt-extracted nuclei suggesting that HMW-DNA fragments represent DNA loop domains (or their associates). Biochemical properties of induced HMW-DNA cleavage suggest involvement of matrix-associated topoisomerase II in excision of DNA loop domains. We demonstrated further that patterns of HMW-DNA cleavage are distinct in plant tissues differed on proliferative activity and differentiation status. The least fragmentation was found in the embryos of dry quiescent seeds, whereas the induction of growth and development was accompanied by increase in HMW-DNA fragmentation. The study of cell nuclei during natural and accelerated ageing of rye seeds demonstrated that loss of germination capacity was associated with decreased excision of chromatin loop domains. Taken together, our data demonstrate that changes in physiological status of plant tissues are accompanied with the changes in patterns of HMW-DNA fragmentation. The HMW-DNA fragments are generated by the scaffold-associated topo II-like endonuclease activity that cleaves nuclear DNA probably at the positions of its attachment to nuclear scaffold. We hypothesize that HMW-DNA cleavage activity is related to some DNA metabolic processes in cell nuclei and is necessary for maintaining active physiological status of plant tissues.