GSBS Student Publications

Title

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

Publication Date

2004-05-26

UMMS Affiliation

Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology; Program in Molecular Medicine

Document Type

Article

Disciplines

Life Sciences | Medicine and Health Sciences

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.

DOI of Published Version

10.1016/j.ymeth.2003.11.016

Source

Methods. 2004 Jul;33(3):213-9. Link to article on publisher's site

Journal/Book/Conference Title

Methods (San Diego, Calif.)

Related Resources

Link to Article in PubMed

PubMed ID

15157888

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