GSBS Student Publications

Title

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

UMMS Affiliation

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

Date

5-26-2004

Document Type

Article

Medical Subject Headings

DNA, Fungal; Schizosaccharomyces; Thymidine

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.

Rights and Permissions

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

DOI of Published Version

10.1016/j.ymeth.2003.11.016

Related Resources

Link to Article in PubMed

Journal Title

Methods (San Diego, Calif.)

PubMed ID

15157888