Mariyana Georgieva

Assessment of DNA strand breaks induced by bleomycin in barley by the comet assay

Comet assay was applied to study induction and repair of DNA damage produced by bleomycin in barley genome. Experimental conditions were adapted to achieve efficient detection of both DNA single‐ and double‐strand breaks. Substantial increase of the parameter “% of DNA in tail” was observed coupled with almost linear dependence from bleomycin concentration, more pronounced for the induction of DNA double‐strand breaks. Data obtained at different recovery periods displayed rapid restoration of breakage, revealing that efficient mechanisms for repair of strand discontinuities induced by bleomycin are functional in barley DNA loop domains. Environ. Mol. Mutagen., 2008.

Molecular cytogenetic characterization of two high protein wheat-Thinopyrum intermedium partial amphiploids

Fluorescence and genomic in situ hybridization (FISH and GISH) were used to establish the cytogenetic constitution of two wheat × Thinopyrum intermedium partial amphiploids H95 and 55(1-57). Both partial amphiploids are high-protein lines having resistance to leaf rust, yellow rust and powdery mildew and have in total 56 chromosomes per cell. Repetitive DNA probes (pTa71, Afa family and pSc119.2) were used to identify the individual wheat chromosomes and to reveal the distribution of these probes within the alien chromosomes. FISH detected 6B tetrasomy in H95 and a null (1D)-tetrasomy (1B) in 55(1-57). GISH was carried out using biotin labeled Th. intermedium DNA and digoxigenin labeled Pseudoroegneria spicata DNA as probes, subsequently. GISH results revealed 44 wheat chromosomes and four Thinopyrum chromosome pairs, including three S and one J chromosome pairs in line H95. Line 55(1-57), contained 42 wheat chromosomes and six Th. intermedium pairs, including two S and one JS pairs. Additionally, two identical translocated chromosome pairs with diminished affinity to the alien chromatin were detected in both amphiploids. Another two translocations were found in 55(1-57), with satellite sections from the Thinopyrum J genome.

DNA damage, repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans

This pilot study was carried out to assess the effect of radio-contaminated Chernobyl environment on plant genome integrity 27 years after the accident. For this purpose, nuclei were isolated from root tips of the soybean seedlings harvested from plants grown in the Chernobyl area for seven generations. Neutral, neutral-alkaline, and methylation-sensitive comet assays were performed to evaluate the induction and repair of primary DNA damage and the epigenetic contribution to stress adaptation mechanisms. An increased level of single and double strand breaks in the radio-contaminated Chernobyl seedlings at the stage of primary root development was detected in comparison to the controls. However, the kinetics of the recovery of DNA breaks of radio-contaminated Chernobyl samples revealed that lesions were efficiently repaired at the stage of cotyledon. Methylation-sensitive comet assay revealed comparable levels in the CCGG methylation pattern between control and radio-contaminated samples with a slight increase of approximately 10% in the latter ones. The obtained preliminary data allow us to speculate about the onset of mechanisms providing an adaptation potential to the accumulated internal irradiation after the Chernobyl accident. Despite the limitations of this study, we showed that comet assay is a sensitive and flexible technique which can be efficiently used for genotoxic screening of plant specimens in natural and human-made radio-contaminated areas, as well as for safety monitoring of agricultural products.

Karyotype reconstruction modulates the sensitivity of barley genome to radiation-induced DNA and chromosomal damage

The potential of cytologically reconstructed barley line D-2946 to cope with the major lesions that hamper genome integrity, namely DNA single- and double-strand breaks was investigated. Strand breaks induced by γ-rays and Li ions were assessed by neutral and alkaline comet assay. Repair capacity after bleomycin treatment was evaluated by agarose gel electrophoresis under neutral and alkaline conditions. Frequencies of radiation-induced chromosome aberrations were also determined. Results indicate that radiation-mediated constitutive rearrangement of the chromosome complement has led to a substantial modulation of the sensitivity of barley genome towards DNA strand breaks, produced by ionising radiation, Li ion implantation and bleomycin in an agent-specific manner, as well as of the clastogenic response to γ-rays. Based on these findings, reconstructed barley karyotype D-2946 can be considered a candidate radio-sensitive line with reduced ability to maintain genome integrity with respect to both DNA and chromosomal damage.

Comparative Analysis of Data Distribution Patterns in Plant Comet Assay

ABSTRACT Screening capabilities of the comet assay have a real potential to study the impact of radiation and different mutagenic sources on induction of damage in DNA in plant nuclei. Heterogeneity of DNA damage data obtained by the application of different agents leads to an inconsistency and variation of the experimental outcomes, obtained after assessment of the comet populations. Although the potential of the comet assay technology has been clearly demonstrated, many important and interesting statistical questions remain. In this respect, different types of data need different statistical designs and this usually makes statistical analysis problematic. We advocate here a greater attention to different classical statistical distributions which best fit to plant comet data. We also demonstrate that all distribution patterns of the % of DNA in tail can be fitted by a Johnson SB distribution.

Molecular Cytogenetic Characterization of a New Reconstructed Barley Karyotype

ABSTRACT Multicolor fluorescence in situ hybridization (FISH) was applied to mitotic metaphase chromosomes of the standard barley variety Freya and a new reconstructed karyotype, PK 88–19, with complete cytological marking of the chromosome complement in order to map physically the rearrangement breakpoints induced by gamma-irradiation. Three repetitive DNA sequences, namely, GAA satellite sequence, Afa-family subclone pAs1, and rDNA clone pTa71, were used as FISH probes. The hybridization sites produced were found to be distributed in specific locations along the chromosomes, thus creating conditions for more detailed cytogenetic characterization of the reconstructed karyotype. The comparative analysis of the distribution patterns of FISH signals in the reconstructed chromosomes resulting from three reciprocal translocations (1H-5H, 2H-7H and 3H-4H) and two pericentric inversions (4H3H and 6H) on the one hand, and the respective standard chromosomes, on the other, allowed a precise identification of the putative regions where the rearrangement breakpoints have occurred. In addition, three new minor rDNA sites located in the long arms of chromosomes 2H, 5H and 6H were identified. Due to the clear morphological distinctions of the different chromosome types and the available detailed positions of their rearrangement breakpoints, PK 88–19 may offer an essential gain in the resolution power over other reconstructed karyotypes in various research areas of barley cytogenetics.