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THYMIDINE UTILIZATION BY tut MUTANTS AND FACILE CLONING OF MUTANT ALLELES BY PLASMID CONVERSION IN S. CEREVISIAE
Robert A. Sclafani 1 and Walton L. Fangman 2
1 Department of Genetics, University of Washington, Seattle,
Washington 98195, and Department of Biochemistry, Biophysics and Genetics,
University of Colorado, Denver, Colorado 80262
2 Department of Genetics, University of Washington, Seattle,
Washington 98195
Plasmid pJM81 contains a Herpes simplex virus thymidine kinase
(TK) gene that is expressed in yeast. Cells containing the plasmid
utilize thymidine (TdR) and the analogue 5-bromodeoxyuridine (BUdR) for specific
incorporation into DNA. TdR auxotrophs, harboring plasmid pJM81 and a mutation
in the yeast gene TMP1 require high concentrations of TdR (300 µg/ml)
to support normal growth rates and the wild-type mitochondrial genome (
+) cannot be maintained. We have identified a yeast gene, TUT1,
in which recessive mutations allow efficient utilization of lower concentrations
of TdR. Strains containing the mutations tmp1 and tut1,
as well as plasmid pJM81, form colonies at 2 µg/ml TdR, grow at nearly
normal rates and maintain the + genome at 50 µg/ml TdR.
These strains can be used to radiolabel DNA specifically and to synchronize
DNA replication by TdR starvation. In addition, the substitution of BUdR for
TdR allows the selective killing of DNA-synthesizing cells by 310-nm irradiation
and allows the separation of replicated and unreplicated forms of DNA by CsCl
equilibrium density banding. We also describe a unique, generally applicable
system for cloning mutant alleles that exploits the fact that Tk+ yeast
cells are sensitive to 5-fluorodeoxyuridine (FUdR) and that gene conversions
can occur between a yeast chromosome and a TK-containing plasmid.
Accepted on July 25, 1986
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