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A number of chemical agents known to influence the key cell cycle regulatory factors were used to assess the requirements of hydroxyurea-treated root meristem cells of Vicia faba for premature condensation of chromosomes (PCC). These included caffeine and 2-aminopurine (inhibitors of ATM/ATR sensor kinases activated by DNA damage or stalled replication forks), inhibitors of protein kinases (staurosporine and wortmannin), inhibitors of protein phosphatases (sodium vanadate and calyculin A), and other compounds like 1,2-dioctyl-sn-glycerol, an activator of protein kinase C, 5-azacytidine, an inhibitor of DNA methyltransferase, dithiothreitol and N-etylmaleimide, capable to up- and down-regulate the activity of Cdc25 phosphatase. Cytological parameters used to evaluate quantitative aspects of PCC allowed us to discriminate various phenotypes of cells and, consistent with the extent of chromosomal fragmentation, to classify them as S- or G2-PCC. Two significant aspects relevant to the induction of premature mitosis in plants seem to emerge: one concerns the inverse relationship between the incidence of mitotic and PCC events, the other refers to the extent with which a variety of chemical agents may activate mechanisms that override the S-M replication checkpoint. 1,2- dioctyl-sn-glycerol, an activator of protein kinase C in animal cells proved extremely effective in stimulation of PCC, in spite of evident lack of molecular targets in plants.
Regardless of the DNA replication stress induced by low concentration of hydroxyurea (HU), root apical meristem cells of Allium cepa keep growing, and some of them override the DNA damage checkpoint mechanisms initiating either premature or an abnormal mitotic chromosome condensation. Prolonged incubation of onion seedlings with HU results in an increased level of immunodetectable proteins sharing epitopes with SUN2, one of the highly conserved elements linking nuclear envelope (NE) to the cytoand nucleoskeletal structures. In addition toNE, phragmoplast and cell plate, our observations extend an array of subcellular compartments at which SUN2-like proteins (SUN2-LPs) are localized. These include cortical preprophase band of microtubules, centromeric regions of ana- and telophase chromosomes, and nuclear bodies (SUN2-NBs) polarly localized in interphase nuclei according to Rabl’s configuration. SUN2- NBs (distinct from fibrillarin-rich Cajal bodies) colocalize with late-replicating areas of heterochromatin and are thought to represent clustered centromeres. Three-dimensional spatial analysis of SUN2-NBs suggests their connections with NE. An enhanced expression and additional localization sites of SUN2-LPs may be correlated with a considerable reprogramming of cellular functions triggered in response to prolonged HU treatment.
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