Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an... more Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an intermediate level of chromosome structure ranging from a few to hundreds of kilobases, a level most relevant for gene regulation. The 3C technique is used to detect physical interactions between sequence elements that are located on the same or on different chromosomes. For instance, physical interactions between distant enhancers and target genes can be measured. The 3C assay uses formaldehyde cross-linking to trap connections between chromatin segments that can, after a number of manipulations, be detected by PCR. This unit describes detailed protocols for performing 3C with yeast Saccharomyces cerevisiae and mammalian cells.
Polycyclic aromatic hydrocarbons (PAHs) are of global environmental concern because they cause ma... more Polycyclic aromatic hydrocarbons (PAHs) are of global environmental concern because they cause many health problems including cancer and inflammation of tissue in humans. Plants are important in removing PAHs from the atmosphere; yet, information on the physiology, cell and molecular biology, and biochemistry of PAH stress responses in plants is lacking. The PAH stress response was studied in Arabidopsis (Arab-idopsis thaliana) exposed to the three-ring aromatic compound, phenanthrene. Morphological symptoms of PAH stress were growth reduction of the root and shoot, deformed trichomes, reduced root hairs, chloro-sis, late flowering, and the appearance of white spots, which later developed into necrotic lesions. At the tissue and cellular levels, plants experienced oxidative stress. This was indicated by localized H 2 O 2 production and cell death, which were detected using 3, 39-diamino-benzidine and trypan blue staining, respectively. Gas chromatography-mass spectrometry and fluorescence spectrometry analyses showed that phenanthrene is internalized by the plant. Gene expression of the cell wall-loosening protein expansin was repressed, whereas gene expression of the pathogenesis related protein PR1 was induced in response to PAH exposure. These findings show that (i) Arabidopsis takes up phenanthrene, suggesting possible degradation in plants, (ii) a PAH response in plants and animals may share similar stress mechanisms, since in animal cells detoxification of PAHs also results in oxidative stress, and (iii) plant specific defence mechanisms contribute to PAH stress response in Arabidopsis.
Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an... more Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an intermediate level of chromosome structure ranging from a few to hundreds of kilobases, a level most relevant for gene regulation. The 3C technique is used to detect physical interactions between sequence elements that are located on the same or on different chromosomes. For instance, physical interactions between distant enhancers and target genes can be measured. The 3C assay uses formaldehyde cross-linking to trap connections between chromatin segments that can, after a number of manipulations, be detected by PCR. This unit describes detailed protocols for performing 3C with yeast Saccharomyces cerevisiae and mammalian cells.
Polycyclic aromatic hydrocarbons (PAHs) are of global environmental concern because they cause ma... more Polycyclic aromatic hydrocarbons (PAHs) are of global environmental concern because they cause many health problems including cancer and inflammation of tissue in humans. Plants are important in removing PAHs from the atmosphere; yet, information on the physiology, cell and molecular biology, and biochemistry of PAH stress responses in plants is lacking. The PAH stress response was studied in Arabidopsis (Arab-idopsis thaliana) exposed to the three-ring aromatic compound, phenanthrene. Morphological symptoms of PAH stress were growth reduction of the root and shoot, deformed trichomes, reduced root hairs, chloro-sis, late flowering, and the appearance of white spots, which later developed into necrotic lesions. At the tissue and cellular levels, plants experienced oxidative stress. This was indicated by localized H 2 O 2 production and cell death, which were detected using 3, 39-diamino-benzidine and trypan blue staining, respectively. Gas chromatography-mass spectrometry and fluorescence spectrometry analyses showed that phenanthrene is internalized by the plant. Gene expression of the cell wall-loosening protein expansin was repressed, whereas gene expression of the pathogenesis related protein PR1 was induced in response to PAH exposure. These findings show that (i) Arabidopsis takes up phenanthrene, suggesting possible degradation in plants, (ii) a PAH response in plants and animals may share similar stress mechanisms, since in animal cells detoxification of PAHs also results in oxidative stress, and (iii) plant specific defence mechanisms contribute to PAH stress response in Arabidopsis.
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Papers by Tomoko Tabuchi