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Mechanisms of nuclear localization of glutathione reductase, subnuclear colocalization with thioredoxin, and genetic analysis of a chemically induced glutathione reductase knockout

Rogers, Lynette K.
http://rave.ohiolink.edu/etdc/view?acc_num=osu1091194762

Abstract Details

2004, Doctor of Philosophy, Ohio State University, Molecular, Cellular, and Developmental Biology.
Glutathione reductase [GR; EC 1.6.4.2] is a ubiquitous, highly conserved protein that has been identified in plants, bacteria, yeast, and mammals. GR expressions and functions in cytosol and mitochondria have been studied, but little is known about mechanisms of import or functions of GR within the nucleus. No classical nuclear localization signal has been identified in GR, but cDNAs containing either mitochondrial targeting signals (MTS) from human MnSOD or from human GR (hGR) target GR transgene products to both the mitochondria and the nucleus. In contrast, cells transfected with a construct coding for a hGR-MTS/GFP fusion protein demonstrated fluorescence only in the mitochondria. Subsequent mutational deletion analyses of the hGR MTS indicated a tight correlation between nuclear and mitochondrial levels of expression and with the numbers of amino acids in the truncated MTSs. The MTS is necessary but not sufficient for nuclear localization of GR. Microscopy studies of cells in culture revealed GR-dependent immunofluorescence in subnuclear clusters. Subsequent investigations indicated that the GR-containing clusters do not colocalize with proteins found in subnuclear structures that exhibit morphologies similar to the anti-GR immunoreactive forms, or with p-histone 3, a protein involved in replication. In both native and transfected cells, GR was colocalized with glutaredoxin, and more strongly with thioredoxin, and colocalization with thioredoxin also exhibited subnuclear clustering. The functions served by the colocalization between GR and TRX protein are not known at this time. A GR-deficient mouse (Neu) was generated by treatment with isopropyl methanesulfonate (a chemical mutagen). RT-PCR analysis indicated that the defect in the GR gene was due to a deletion. Using PCR and Southern blot strategies, the deletion was isolated to the region between intron 1 and intron 5. Sequence analysis of a PCR product that included the putative deletion identified the precise breakpoints of the deletion and indicated that the Neu mice have a 12.8 kb deletion in their genomic GR gene and are a genetic knockout. The Neu mice are surprisingly healthy despite the absence of a gene so highly conserved across such a wide range of life forms.
Charles Smith (Advisor)
148 p.
glutathione reductase; glutathione; nuclear localization; mitochondrial localization; thioredoxin; glutathione reductase knockout mouse

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Citations

  • Rogers, L. K. (2004). Mechanisms of nuclear localization of glutathione reductase, subnuclear colocalization with thioredoxin, and genetic analysis of a chemically induced glutathione reductase knockout [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1091194762

    APA Style (7th edition)

  • Rogers, Lynette. Mechanisms of nuclear localization of glutathione reductase, subnuclear colocalization with thioredoxin, and genetic analysis of a chemically induced glutathione reductase knockout. 2004. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1091194762.

    MLA Style (8th edition)

  • Rogers, Lynette. "Mechanisms of nuclear localization of glutathione reductase, subnuclear colocalization with thioredoxin, and genetic analysis of a chemically induced glutathione reductase knockout." Doctoral dissertation, Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1091194762

    Chicago Manual of Style (17th edition)

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