Thymidine utilization by tut mutants and facile cloning of mutant alleles by plasmid conversion in S. cerevisiae - PubMed (original) (raw)
Thymidine utilization by tut mutants and facile cloning of mutant alleles by plasmid conversion in S. cerevisiae
R A Sclafani et al. Genetics. 1986 Nov.
Abstract
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 micrograms/ml) to support normal growth rates and the wild-type mitochondrial genome (rho+) 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 micrograms/ml TdR, grow at nearly normal rates and maintain the rho+ genome at 50 micrograms/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.
Similar articles
- Expression of the Herpes simplex virus thymidine kinase gene in Saccharomyces cerevisiae.
McNeil JB, Friesen JD. McNeil JB, et al. Mol Gen Genet. 1981;184(3):386-93. doi: 10.1007/BF00352510. Mol Gen Genet. 1981. PMID: 6278252 - The herpes simplex virus thymidine kinase gene is not transcribed in Saccharomyces cerevisiae.
Kiss GB, Pearlman RE, Cornish KV, Friesen JD, Chan VL. Kiss GB, et al. J Bacteriol. 1982 Feb;149(2):542-7. doi: 10.1128/jb.149.2.542-547.1982. J Bacteriol. 1982. PMID: 6276360 Free PMC article. - Control of Herpes simplex virus thymidine kinase gene expression in Saccharomyces cerevisiae by a yeast promoter sequence.
Zhu XL, Ward C, Weissbach A. Zhu XL, et al. Mol Gen Genet. 1984;194(1-2):31-41. doi: 10.1007/BF00383493. Mol Gen Genet. 1984. PMID: 6328218 - Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK.
Sclafani RA, Fangman WL. Sclafani RA, et al. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5821-5. doi: 10.1073/pnas.81.18.5821. Proc Natl Acad Sci U S A. 1984. PMID: 6091111 Free PMC article. - Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids.
Kit S, Otsuka H, Qavi H, Hazen M. Kit S, et al. Proc Natl Acad Sci U S A. 1981 Jan;78(1):582-6. doi: 10.1073/pnas.78.1.582. Proc Natl Acad Sci U S A. 1981. PMID: 6264449 Free PMC article.
Cited by
- Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.
Burke DJ, Church D. Burke DJ, et al. Mol Cell Biol. 1991 Jul;11(7):3691-8. doi: 10.1128/mcb.11.7.3691-3698.1991. Mol Cell Biol. 1991. PMID: 2046672 Free PMC article. - G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover.
Salama SR, Hendricks KB, Thorner J. Salama SR, et al. Mol Cell Biol. 1994 Dec;14(12):7953-66. doi: 10.1128/mcb.14.12.7953-7966.1994. Mol Cell Biol. 1994. PMID: 7969135 Free PMC article. - DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase.
Hollingsworth RE Jr, Sclafani RA. Hollingsworth RE Jr, et al. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6272-6. doi: 10.1073/pnas.87.16.6272. Proc Natl Acad Sci U S A. 1990. PMID: 2166954 Free PMC article. - Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.
Jackson AL, Pahl PM, Harrison K, Rosamond J, Sclafani RA. Jackson AL, et al. Mol Cell Biol. 1993 May;13(5):2899-908. doi: 10.1128/mcb.13.5.2899-2908.1993. Mol Cell Biol. 1993. PMID: 8474449 Free PMC article. - Unbiased segregation of yeast chromatids in Saccharomyces cerevisiae.
Burke DJ. Burke DJ. Chromosome Res. 2013 May;21(3):193-202. doi: 10.1007/s10577-013-9348-x. Chromosome Res. 2013. PMID: 23681653 Review.
References
- J Bacteriol. 1967 May;93(5):1662-70 - PubMed
- J Gen Microbiol. 1968 Dec;54(2):307-17 - PubMed
- Eur J Biochem. 1969 Dec;11(2):249-60 - PubMed
- Mol Gen Genet. 1970;109(2):107-22 - PubMed
- Exp Cell Res. 1971 Aug;67(2):389-401 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases