In vivo labeling of fission yeast DNA with thymidine and thymidine analogs
Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology; Program in Molecular Medicine
Life Sciences | Medicine and Health Sciences
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.
DOI of Published Version
Methods. 2004 Jul;33(3):213-9. Link to article on publisher's site
Methods (San Diego, Calif.)
Sivakumar S, Porter-Goff ME, Patel PK, Benoit K, Rhind NR. (2004). In vivo labeling of fission yeast DNA with thymidine and thymidine analogs. GSBS Student Publications. https://doi.org/10.1016/j.ymeth.2003.11.016. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/1121