Detekcija genotoksičnosti s pomoću biljnih biotestova - molekularno-citogenetički pristup

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay).Za prevenciju oštećivanja molekule DNA zbog onečišćenja okoliša nužno je razumijevanje bioloških posljedica nastalih oštećenjem DNA i molekularnih mehanizama djelovanja genotoksikanata koji vode ili popravku ili promjenama genskog materijala. Do danas je usavršen niz testova za detekciju genotoksičnog djelovanja koji omogućuju identifikaciju supstancija koje reagiraju s molekulom DNA. Raspoloživi podaci pokazuju da su biljni biotestovi vrlo važni u detekciji genotoksičnog onečišćenja okoliša, kao i za uspostavljanje nadzornih sustava u okolišu. Biljni test-sustav može otkriti široki raspon genskog oštećenja uključujući mutacije gena i kromosomske aberacije. Nove molekularno-citogenetičke metode omogućuju analizu genotoksičnog djelovanja na razini kromosoma i molekule DNA. Metoda FISH-a ("fluorescent in situ hybridization") pruža nove mogućnosti za detekciju i analizu kromosomskih preraspodjela. Lomovi u molekuli DNA mogu se uspješno detektirati s pomoću metode TUNEL i gel-elektroforeze pojedinačnih stanica (komet test)

Detekcija genotoksičnosti s pomoću biljnih biotestova - molekularno-citogenetički pristup

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay).Za prevenciju oštećivanja molekule DNA zbog onečišćenja okoliša nužno je razumijevanje bioloških posljedica nastalih oštećenjem DNA i molekularnih mehanizama djelovanja genotoksikanata koji vode ili popravku ili promjenama genskog materijala. Do danas je usavršen niz testova za detekciju genotoksičnog djelovanja koji omogućuju identifikaciju supstancija koje reagiraju s molekulom DNA. Raspoloživi podaci pokazuju da su biljni biotestovi vrlo važni u detekciji genotoksičnog onečišćenja okoliša, kao i za uspostavljanje nadzornih sustava u okolišu. Biljni test-sustav može otkriti široki raspon genskog oštećenja uključujući mutacije gena i kromosomske aberacije. Nove molekularno-citogenetičke metode omogućuju analizu genotoksičnog djelovanja na razini kromosoma i molekule DNA. Metoda FISH-a ("fluorescent in situ hybridization") pruža nove mogućnosti za detekciju i analizu kromosomskih preraspodjela. Lomovi u molekuli DNA mogu se uspješno detektirati s pomoću metode TUNEL i gel-elektroforeze pojedinačnih stanica (komet test)

DNA damage induced by mutagens in plant and human cell nuclei in acellular comet assay.

Higher plant cells have a long tradition of use in the studies on environmental mutagenesis in situ, especially in relation to human health risk determination. The studies on the response of plant and human cells to physical and chemical mutagens showed differences in their sensitivity. The differences in the presence of cell components in plants and humans could influence such response. Additionally, the level of the organization of the employed material could influence DNA-damaging effect: leukocytes are isolated cells and plant--an intact organism. To preclude these obstacles, the effects of direct treatment of isolated nuclei with genotoxic agents were determined to compare the sensitivity of plant and human cells. In the present study, we have determined the DNA-damaging effects of two chemical mutagens: maleic acid hydrazide (MH) and N-methyl-N-nitroso-urea (MNU) applied to isolated nuclei of both plant and human cells. In order to compare the sensitivity of the nuclei of Nicotiana tabacum var. xanthi and the nuclei of leukocytes, the acellular Comet assay was carried out. The results showed higher sensitivity of the nuclei of leukocytes as compared to the nuclei of plant cells to mutagenic treatment with the applied doses of MH and MNU

Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna

Background: Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. Results: Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor. Conclusions: The obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms

Comparative Analysis of rDNA Distribution in Chromosomes of Various Species of Brassicaceae

• Background and Aims The Brassicaceae family encompasses numerous species of great agronomic importance, belonging to such genera, as Brassica, Raphanus, Sinapis and Armoracia. Many of them are characterized by extensive intraspecific diversity of phenotypes. The present study focuses on the polymorphism of number, appearance and chromosomal localization of ribosomal DNA (rDNA) sites and, when possible, in relation to polyploidy, in 42 accessions of Brassica species and ten accessions of Diplotaxis, Eruca, Raphanus and Sinapis species

Additional file 1: Figure S1. of Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna

Localization of restriction endonucleases recognition sites (B: Bam HI, EI: Eco RI, EV: Eco RV, S: Sph I, Xb: Xba I, Xh: Xho I) in the coding region (a) and IGS (b) of Atropa belladonna 45S rDNA. (JPG 2102 kb

Additional file 7: Figure S7. of Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna

Nucleotide sequence comparison of 5′ (a) and 3′ (b) portions of 5S rDNA coding region of Atropa belladonna (Abel: this study, consensus sequence – see Fig. 5), Solanum lycopersicum (Slyc: Genbank Acc. No X55697), S. tuberosum (Stub: X82780), S. pinnatisectum (Spnt: X82779) and Petunia hybrida (Phyb-A: X07930, Phyb-B: X07929, Phyb-C: X07928). Sequences of primers Pr5S-R (A) and Pr5S-L (B) used for A. belladonna 5S rDNA amplification are marked as arrows. (JPG 1600 kb