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

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 growt...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2004-07, Vol.33 (3), p.213-219
Main Authors: Sivakumar, Sasirekha, Porter-Goff, Mary, Patel, Prasanta K., Benoit, Kristen, Rhind, Nicholas
Format: Article
Language:English
Subjects:
BrdU
CldU
DNA labeling
DNA, Fungal - genetics
DNA, Fungal - metabolism
Fission yeast
Human equilibrative nucleoside transporter 1
IdU
Schizosaccharomyces - genetics
Schizosaccharomyces - metabolism
Schizosaccharomyces pombe
Thymidine - analogs & derivatives
Thymidine - genetics
Thymidine - metabolism
Thymidine incorporation
Thymidine kinase
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Summary: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.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2003.11.016