FISH in analysis of gamma ray-induced micronuclei formation in barley

Abstract

A micronucleus test in combination with fluorescent in situ hybridization (FISH) using telomere-, centromere-specific probes and 5S and 25S rDNA was used for a detailed analysis of the effects of gamma ray irradiation on the root tip meristem cells of barley, Hordeum vulgare (2n = 14). FISH with four DNA probes was used to examine the involvement of specific chromosomes or chromosome fragments in gamma ray-induced micronuclei formation and then to explain their origin. Additionally, a comparison of the possible origin of the micronuclei induced by physical and chemical treatment: maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU) was done. The micronuclei induced by gamma ray could originate from acentric fragments after chromosome breakage or from whole lagging chromosomes as a result of a dysfunction of the mitotic apparatus. No micronuclei containing only centromeric signals were found. An application of rDNA as probes allowed it to be stated that 5S rDNA–bearing chromosomes are involved in micronuclei formation more often than NOR chromosomes. This work allowed the origin of physically- and chemically-induced micronuclei in barley cells to be compared: the origin of micronuclei was most often from terminal fragments. FISH confirmed its usefulness in the characterization of micronuclei content, as well as in understanding and comparing the mechanisms of the actions of mutagens applied in plant genotoxicity.

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

“…These observations indicate that despite their similar size, Bd4 chromosomes are involved in the MH-induced micronuclei formation more often than Bd5 chromosomes. Similar results were observed in gamma ray-induced micronuclei in barley [4]. Moreover, an additional locus of 35S rDNA was present after MH treatment in barley cells, which suggests that either a duplication or double strand breaks (DBS) occurred within the 35S rDNA locus.…”

“…Our data correspond to that reported by Jovtchev et al [1], who showed that only 1% of MNU-induced barley micronuclei revealed centromere DNA signals. Interestingly, the frequency of such micronuclei in barley that was subjected to MH treatment was considerably higher (6%) [5], whereas they were not observed at all in barley cells after gamma radiation [4]. Such discrepancies in the frequencies may indicate that only MH and MNU cause interstitial chromosome breaks.…”

“…Additionally, when FISH is used with the micronuclei (MN) test, it provides a better understanding of the micronuclei origin [3]. The effects of various mutagens have been analyzed in large-genome grass species, such as Hordeum vulgare (barley), in order to determine the origin of both physically [4] and chemically induced [1, 5] micronuclei. Similar studies have also been performed after maleic hydrazide (MH) treatment in a long-term in vitro root culture of Crepis capillaris , which is a weedy representative of Asteraceae [3].…”

Genomic Organization Under Different Environmental Conditions:Hoplosternum Littoraleas a Model

“…The variable micronuclei compositions also are observed in barley (Hordeum vulgare) root cells to analyze the effects of Gamma ray irradiation. 67 Furthermore, the presence of 18S rDNA in micronuclei of tamoatás may result in problems in the production of ribosomes, which are responsible for protein synthesis, since the 18S rDNA was shown to be active in micronuclei when subjected to impregnation with silver nitrate.…”

Molecular cytogenetic characterisation of Salix viminalis L. using repetitive DNA sequences

“…The probe pTA794 contains a 410-bp BamHI fragment including an intergenic spacer and is part of the 5S rDNA gene family isolated from wheat (Gerlach and Dyer 1980). The HT100.3 telomere repeat (TTTAGGG) n sequences were originally isolated and amplified from Arabidopsis thaliana L. (Juchimiuk-Kwasniewska et al 2011). The pAl1 178 bp tandem repeat was cloned from Arabidopsis thaliana L. (Martinez-Zapater et al 1986).…”