Abstract
To investigate the chromosome dynamics during mitosis, it is convenient to mark the discrete chromosome foci and then analyze their spatial rearrangements during prophase condensation and telophase decondensation. To label the chromosome regions in plant chromosomes, we incorporated the synthetic nucleotide, 5-ethynyl-2’-deoxyuridine (EdU), which can be detected by click-chemistry, into chromatin during replication. Here, we described a protocol of a method based on the application of semi-thin sections of Nigella damascena L. roots embedded in LR White acrylic resin. The thickness of semi-thin (100-250 nm) sections is significantly lower than that of optical sections even if a confocal microscope was used. This approach may also be suitable for work with any tissue fragments or large cells (oocytes, cells with polytene chromosomes, etc.).
Keywords: Plant, Chromosome, Replication, EdU, Click-chemistry
Background
Most data concerning chromosome organization have been acquired from studies of a small number of model organisms, the majority of which are mammals. In plants with large genomes, the chromosomes are significantly larger than the animal chromosomes that have been studied to date. To investigate the chromosome dynamics during mitosis, it is necessary to mark the discrete chromosome foci and then analyze their spatial rearrangements during prophase condensation and telophase decondensation. To label the chromosome regions, we incorporated the synthetic nucleotide, 5-ethynyl-2’-deoxyuridine (EdU) (Kuznetsova et al., 2017). Detection of EdU is based on a click-reaction, which is a copper catalyzed reaction between an azide and an alkyne. The EdU contains the alkyne which can react with the azide-containing detection reagent.The most suitable distribution of labeled regions (i.e., separated labeled dots) was seen in cells which incorporated EdU during late S-phase. The brief pulse labeled all S-phase cells, and the initial appearance of EdU-labeled mitotic figures thus denoted the time needed for cells labeled in late S-phase to traverse into mitosis.Root apical meristem does not allow for the acquisition of high-resolution images because of the out-of-focus fluorescence. Here, we described a protocol of a method based on the application of semi-thin (100-250 nm) sections of roots embedded in LR White acrylic resin. LR White is a polyhydroxy-aromatic acrylic resin with low toxicity and ultra-low viscosity. The polymerized resin is hydrophilic (sections freely permeable to aqueous solutions). The thickness of semi-thin sections is significantly lower than that of optical sections even if a confocal microscope was used. This approach may also be suitable for work with any tissue fragments or large cells (e.g., cells with polytene chromosomes).
Materials and Reagents
Equipment
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Notes
Recipes
Acknowledgments
The work was supported by the Russian Science Foundation (project 14-15-00199). The protocol was adapted from previous work (Kuznetsova et al., 2017). The authors declare no conflicts of interest or competing interests that may impact the design and implementation of this protocol.
References
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