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    In vivo labeling of fission yeast DNA with thymidine and thymidine analogs

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    Authors
    Sivakumar, Sasirekha
    Porter-Goff, Mary Elizabeth
    Patel, Prasanta K.
    Benoit, Kristen
    Rhind, Nicholas R.
    UMass Chan Affiliations
    Program in Molecular Medicine
    Department of Biochemistry and Molecular Pharmacology
    Graduate School of Biomedical Sciences
    Document Type
    Journal Article
    Publication Date
    2004-05-26
    Keywords
    DNA, Fungal; Schizosaccharomyces; Thymidine
    Life Sciences
    Medicine and Health Sciences
    
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    Link to Full Text
    http://dx.doi.org/10.1016/j.ymeth.2003.11.016
    Abstract
    In vivo labeling of DNA with thymidine and thymidine analogs has long been a cornerstone of replication studies. Unfortunately, yeast lack a thymidine salvage pathway and thus do not incorporate exogenous thymidine. Specifically, yeast neither efficiently take up exogenous thymidine from their growth media nor phosphorylate it to thymidylate, the precursor of dTTP. We have overcome these problems in fission yeast by expressing the human equilibrative nucleoside transporter 1 (hENT1) along with herpes simplex virus thymidine kinase (tk). hENT1 tk cells are healthy and efficiently incorporate exogenous thymidine and thymidine analogs. We present protocols for labeling DNA with tritiated thymidine, for in situ detection of incorporated BrdU by immunofluorescence, for double labeling with CldU and IdU, for CsCl gradient separation of IdU-labeled DNA, and for using hENT1 and tk as both positive and negative selection markers.
    Source
    Methods. 2004 Jul;33(3):213-9. Link to article on publisher's site
    DOI
    10.1016/j.ymeth.2003.11.016
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/32558
    PubMed ID
    15157888
    Related Resources
    Link to Article in PubMed
    ae974a485f413a2113503eed53cd6c53
    10.1016/j.ymeth.2003.11.016
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