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A reliable and sensitive technique for detection of DNA damage is the Single Cell Gel Assay (also known as comet assay). In this procedure, single cells are exposed to an electric field and damaged DNA moves out of the nucleus, which can be visualized by ethidium bromide staining. Cells display a comet appearance under fluorescence microscopy, the size of the DNA “tail” of the comet being in direct correlation with the extension of DNA damage. In this work we have optimized the comet assay using Saccharomyces cerevisiae cells in order to assess DNA damage caused by oxidative stress. We show that genomic DNA damage increases as the yeast population enters the post-diauxic phase. In addition, the tail comet length decreases by incubation of yeast cells with natural compounds, known to protect cells against oxidative stress (quercetin, ur...
Ceramides are naturally occurring sphingolipids that cause several biological effects, including induction of cell cycle arrest, cell maturation, terminal cell differentiation, cell senescence, and cell death. It is well established that ceramide induces both apoptosis and autophagy in mammalian cells. However, the molecular mechanisms induced by ceramide in yeast, as well as the signalling systems involved, are poorly understood. It is known that phytoceramides, assumed to be the yeast counterpart of the mammalian ceramides, mediate regulation of cell growth and stress responses in yeast. However, their role has proved difficult to define. In this study, we characterized the effect of exogenous N-Acetyl-phytosphingosine (C2-phytoceramide) on Saccharomyces cerevisiae W303 1A cells. We found that C2-phytoceramide induced death in a dos...
Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and ox...
This work was funded by FEDER through the program “Programa Operacional Factores de Competitividade-COMPETE” and by FCT through the projects Pest-C/BIA/UI4050/2011 and FCOMP-01-0124-FEDER-007047. F.A. was supported by an FCT fellowship (FCOMP-01-0124-FEDER-007047).
A reliable and sensitive technique for detection of DNA damage is the Single Cell Gel Assay (comet assay). In this procedure, cells are exposed to an electric field and damaged DNA moves out of the nucleus, which can be visualized by ethidium bromide staining, displaying a comet appearance under fluorescence microscopy. We show that there is a correlation between peroxide concentration and comet tail length and that DNA damage increases as the yeast population enters the post-diauxic phase, which is in agreement with the accumulation of DNA damage with aging. We show also that quercetin, an antigenotoxic natural compound, and a water extract from Ginkgo biloba leaves protect DNA against oxidative stress. DNA repair kinetics was assessed as well by the comet assay after oxidative challenge and a subsequent recovery period. For t...
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