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dc.contributor.authorSivakumar, Sasirekha
dc.contributor.authorPorter-Goff, Mary Elizabeth
dc.contributor.authorPatel, Prasanta K.
dc.contributor.authorBenoit, Kristen
dc.contributor.authorRhind, Nicholas R.
dc.date2022-08-11T08:08:48.000
dc.date.accessioned2022-08-23T16:08:53Z
dc.date.available2022-08-23T16:08:53Z
dc.date.issued2004-05-26
dc.date.submitted2008-12-11
dc.identifier.citationMethods. 2004 Jul;33(3):213-9. <a href="http://dx.doi.org/10.1016/j.ymeth.2003.11.016">Link to article on publisher's site</a>
dc.identifier.issn1046-2023 (Print)
dc.identifier.doi10.1016/j.ymeth.2003.11.016
dc.identifier.pmid15157888
dc.identifier.urihttp://hdl.handle.net/20.500.14038/32558
dc.description.abstractIn 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.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=15157888&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://dx.doi.org/10.1016/j.ymeth.2003.11.016
dc.subjectDNA, Fungal; Schizosaccharomyces; Thymidine
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.titleIn vivo labeling of fission yeast DNA with thymidine and thymidine analogs
dc.typeJournal Article
dc.source.journaltitleMethods (San Diego, Calif.)
dc.source.volume33
dc.source.issue3
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_sp/1121
dc.identifier.contextkey680294
html.description.abstract<p>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.</p>
dc.identifier.submissionpathgsbs_sp/1121
dc.contributor.departmentProgram in Molecular Medicine
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.contributor.departmentGraduate School of Biomedical Sciences
dc.source.pages213-9


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