Detecting Brachypodium distachyon Chromosomes Bd4 and Bd5 in MH- and X-Ray-Induced Micronuclei Using mcFISH.

Micronuclei are biomarkers of genotoxic effects and chromosomal instability. They are formed when chromosome fragments or whole chromosomes fail to disjoin into daughter nuclei. We present qualitative and quantitative analyses of the involvement of specific chromosome regions of chromosomes Bd4 and Bd5 in the formation of micronuclei of Brachypodium distachyon root tip cells following maleic hydrazide (MH) treatment and X-radiation. This is visualised by cytomolecular approaches using bacterial artificial chromosome (BAC)-based multicolour fluorescence in situ hybridisation (mcFISH) in combination with 5S and 25S rDNA probes. The results showed that the long arm of submetacentric chromosome Bd4 forms micronuclei at twice the frequency of its short arm, suggesting that the former is more prone to double-strand breaks (DSBs). In contrast, no difference was observed in the frequency of micronuclei derived from the long and short arms of submetacentric chromosome Bd5. Interestingly, the proximal region of the short arm of Bd5 is more prone to DSBs than its distal part. This demonstrates that 5S rDNA and 35S rDNA loci are not "hot spots" for DNA breaks after the application of these mutagens.

Dissecting the chromosomal composition of mutagen-induced micronuclei in Brachypodium distachyon using multicolour FISH.

Background and Aims: Brachypodium distachyon (Brachypodium) is a model species for temperate cereals and other economically important grasses. Its favourable cytogenetic features and advanced molecular infrastructure make it a good model for understanding the mechanisms of instability of plant genomes after mutagenic treatment. The aim of this study was to qualitatively and quantitatively assess the composition and origin of micronuclei arising from genomic fracture, and to detect possible 'hot spots' for mutagen-induced DNA breaks. Methods: Seeds of Brachypodium were treated with maleic hydrazide (MH) or X-rays. The structure of mutagen-induced micronuclei was analysed in root-tip meristematic cells using multicolour fluorescence in situ hybridization (mcFISH) with various repetitive (5S rDNA, 25S rDNA, telomeric, centromeric) and low-repeat [small and large pools of bacterial artificial chromosome (BAC) clones specific for chromosome Bd1] DNA sequences. Key Results: The majority of micronuclei derive from large, acentric fragments. X-rays caused more interstitial DNA breaks than MH. Double-strand breaks rarely occurred in distal chromosome regions. Bd1 contributed to the formation of more mutagen-induced micronuclei than expected from random chromosome involvement. Conclusions: mcFISH with chromosome-specific BAC clones offers insight into micronuclei composition, in so far as it allows their origin and formation to be determined more specifically. A reliable assay for micronuclei composition is crucial for the development of modern genotoxicity tests using plant cells. The combination of mutagenic treatments and well-developed cytomolecular resources in Brachypodium make this model species very promising for plant mutagenesis research.

Impact of Mutagens on DNA Replication in Barley Chromosomes.

Replication errors that are caused by mutagens are critical for living cells. The aim of the study was to analyze the distribution of a DNA replication pattern on chromosomes of the H. vulgare 'Start' variety using pulse 5-ethynyl-2'-deoxyuridine (EdU) labeling, as well as its relationship to the DNA damage that is induced by mutagenic treatment with maleic hydrazide (MH) and γ ray. To the best of our knowledge, this is the first example of a study of the effects of mutagens on the DNA replication pattern in chromosomes, as well as the first to use EdU labeling for these purposes. The duration of the cell cycle of the Hordeum vulgare 'Start' variety was estimated for the first time, as well as the influence of MH and γ ray on it. The distribution of the signals of DNA replication along the chromosomes revealed relationships between DNA replication, the chromatin structure, and DNA damage. MH has a stronger impact on replication than γ ray. Application of EdU seems to be promising for precise analyses of cell cycle disturbances in the future, especially in plant species with small genomes.

Brachypodium distachyon - A Useful Model in the Qualification of Mutagen-Induced Micronuclei Using Multicolor FISH.

Brachypodium distachyon (Brachypodium) is now intensively utilized as a model grass species in various biological studies. Its favorable cytological features create a unique foundation for a convenient system in mutagenesis, thereby potentially enabling the 'hot spots' and 'cold spots' of DNA damage in its genome to be analyzed. The aim of this study was to analyze the involvement of 5S rDNA, 25S rDNA, the Arabidopsis-type (TTTAGGG)n telomeric sequence and the Brachypodium-originated centromeric BAC clone CB33J12 in the micronuclei formation in Brachypodium root tip cells that were subjected to the chemical clastogenic agent maleic hydrazide (MH). To the best of our knowledge, this is the first use of a multicolor fluorescence in situ hybridization (mFISH) with four different DNA probes being used simultaneously to study plant mutagenesis. A quantitative analysis allowed ten types of micronuclei, which were characterized by the presence or absence of specific FISH signal(s), to be distinguished, thus enabling some specific rules governing the composition of the MH-induced micronuclei with the majority of them originating from the terminal regions of chromosomes, to be identified. The application of rDNA sequences as probes showed that 5S rDNA-bearing chromosomes are involved in micronuclei formation more frequently than the 25S rDNA-bearing chromosomes. These findings demonstrate the promising potential of Brachypodium to be a useful model organism to analyze the effects of various genotoxic agents on the plant nuclear genome stability, especially when the complex FISH-based and chromosome-specific approaches such as chromosome barcoding and chromosome painting will be applied in future studies.

DNA replication after mutagenic treatment in Hordeum vulgare.

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed.