Isolation and characterization of yeast mutants auxotrophic for 2′-deoxythymidine 5′-monophosphate

Little, Joseph W.; Haynes, Robert H.

doi:10.1007/bf00431440

Cited by 68 publications

(41 citation statements)

References 33 publications

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“…This media was lethal to a wild-type strain but supported growth of an isogenic TK-hENT1 strain. However, consistent with previous results (Little and Haynes 1979), when we used this media to select for TK transformants, most of the colonies recovered were r 2 petites that lacked respiratory function. Replacing glucose in the media with the nonfermentable carbon source glycerol (YPGly +AF) allowed selection of cells that retained functional mitochondrial genomes ( Figure S2).…”

Section: Resultssupporting

confidence: 90%

“…Thymidine kinase from human herpes simplex virus (HSV-TK) was first expressed in yeast in the early 1980s (McNeil and Friesen 1981). Whether thymidylate synthase was inhibited by antifolates or a cdc21 (tmp1) mutation, a high frequency of r 2 petites lacking mitochondrial function were generated (Little and Haynes 1979). The tut1 genetic background was shown to exhibit enhanced thymidine uptake, which allowed the propagation of 90% r + cdc21 cells on glucose and the development of a counterselection scheme to replace plasmid TK with a captured piece of nuclear DNA (Sclafani and Fangman 1986).…”

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High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces

2014

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Current genome editing techniques available for Saccharomyces yeast species rely on auxotrophic markers, limiting their use in wild and industrial strains and species. Taking advantage of the ancient loss of thymidine kinase in the fungal kingdom, we have developed the herpes simplex virus thymidine kinase gene as a selectable and counterselectable marker that forms the core of novel genome engineering tools called the Haploid Engineering and Replacement Protocol (HERP) cassettes. Here we show that these cassettes allow a researcher to rapidly generate heterogeneous populations of cells with thousands of independent chromosomal allele replacements using mixed PCR products. We further show that the high efficiency of this approach enables the simultaneous replacement of both alleles in diploid cells. Using these new techniques, many of the most powerful yeast genetic manipulation strategies are now available in wild, industrial, and other prototrophic strains from across the diverse Saccharomyces genus.G ENOME editing is a precise and powerful tool to investigate basic genetic processes or to reprogram an organism's metabolism. Techniques to precisely manipulate genomes exist for many model organisms (Storici et al. 2003;Gratz et al. 2013;Hwang et al. 2013;Jiang et al. 2013;Tzur et al. 2013), but not all features of these approaches are easily portable to closely related species. The genus Saccharomyces is highly experimentally tractable, and laboratory strains of all seven natural species can be genetically manipulated Liti et al. 2013). Saccharomyces cerevisiae is the most well-known member of the genus due to its role in brewing (Lodolo et al. 2008), biofuels (Steen et al. 2008;Matsushika et al. 2009), winemaking (Peris et al. 2012, and baking, as well as a model system for the biological sciences (Botstein and Fink 2011). Other members of the genus are also used by humans in the form of interspecies hybrids, such as the S. cerevisiae 3 Saccharomyces kudriavzevii hybrids used to ferment some wines and Belgian beers (Peris et al. 2012) and the S. cerevisiae 3 Saccharomyces eubayanus (Libkind et al. 2011) hybrids found in the brewing of lager-style beers around the world. The Saccharomyces genus is also an emerging "model genus" for molecular evolution, and several experimentally tractable species are now used routinely in evolutionary genetics research (Hittinger 2013). Efficient genome editing of these diverse Saccharomyces yeasts would therefore provide new avenues of investigation for basic and applied research.One major reason for the popularity of S. cerevisiae as a model system is the availability of powerful genetic manipulation tools. One of these tools is the URA3 selection/counterselection system (Boeke et al. 1984). URA3 is a gene required for the de novo synthesis of uracil. Thus, the URA3 gene can be used as a selectable marker in ura3 strains by selecting for the ability to grow on synthetic media without uracil. The deactivation or replacement of URA3 can also be selected for using synt...

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Section: Resultssupporting

confidence: 90%

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High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces

2014

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“…S. cerevisiae GY712, provided by Gareth R. Taylor, Mt. Sinai Research Institute, Toronto, Ontario, Canada, was grown at 34°C in YPD broth (17) supplemented with 1.5 mg of KH2PO4 per ml and 100 ,ug of dTMP per ml. This strain is Tup-and thus permeable to exogenous dTMP (35).…”

Section: Methodsmentioning

confidence: 99%

Cloning, purification, and properties of Candida albicans thymidylate synthase

et al. 1989

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The thymidylate synthase (TS) gene was isolated from a genomic Candida albicans library by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was localized on a 4-kilobase HindIll DNA fragment and was shown to be expressed in a Thy-strain of Escherichia coi. The Thymidylate synthase (TS) has been characterized from a wide variety of organisms and is a proven target in cancer chemotherapy (25). TS should be a good chemotherapeutic target in Candida albicans, a common fungal pathogen, since the product of the enzyme, dTMP, can only be synthesized de novo in yeasts; they lack thymidine kinase and are impermeable to thymine, thymidine, and dTMP (6). Effective inhibition of TS in yeasts would result in death, since these organisms are unable to produce their own dTMP or obtain it from the environment. 5-Fluorocytosine inhibits C. albicans and several other fungi both in vitro and in vivo (3). Furthermore, treatment of C. albicans with 5-fluorocytosine (9) results in 5-fluoro-dUMP accumulation and inhibition of TS, thus implicating this enzyme as a chemotherapeutic target in fungi.Information obtained from the characterization of target enzymes in vitro has contributed to the design of new potential chemotherapeutic agents. Such studies are facilitated by the availability of large quantities of pure enzyme. Since low levels of TS are present in cultures of C. albicans, the cloning and overexpression of C. albicans TS in Escherichia coli was undertaken. We report the isolation of the C. albicans TS gene by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was sequenced with about 400 base pairs (bp) of DNA 5' to the gene and a short 3'-flanking region and expressed in E. coli by using a T7 expression vector. The purified enzyme from both C. albicans and E. coli was prepared, and its properties were examined to ensure that the cloned TS enzyme as expressed in E. coli was identical to the native TS enzyme of C. albicans. MATERIALS AND METHODSStrains, plasmids, and media. isolated as a pool of 8-to 10-kilobase-pair (kb) fragments, into the BamHI site of the low-copy-number S. cerevisiae-E. coli shuttle vector pYSK35 (Fig. 1). The S. cerevisiae-E. coli shuttle vector YEp13 (8)

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“…Since S. cerevisiae lacks thymidine kinase (14), dTMP is synthesized only by the de novo pathway in which methylenetetrahydrofolate, which is required for the conversion of dUMP to dTMP, is synthesized from dihydrofolate via tetrahydrofolate. Dihydrofolate reductase (DHFR), the enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate, is inhibited by aminopterin or methotrexate (9,19,32). S. cerevisiae is susceptible to these drugs when the de novo synthesis of dihydrofolate is blocked by sulfanilamide (9,19,32), a compound which inhibits conversion of hydroxymethyldihydropterin pyrophosphate to dihydropterin (Fig.…”

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confidence: 99%

“…Dihydrofolate reductase (DHFR), the enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate, is inhibited by aminopterin or methotrexate (9,19,32). S. cerevisiae is susceptible to these drugs when the de novo synthesis of dihydrofolate is blocked by sulfanilamide (9,19,32), a compound which inhibits conversion of hydroxymethyldihydropterin pyrophosphate to dihydropterin (Fig. 1) Denhardt solution (lx Denhardt solution is 0.02% [wt/vol] each of bovine serum albumin, Ficoll, and polyvinyl pyrrolidone) and 100 ,ug of sonicated salmon sperm DNA per ml at 42°C; filters were washed with 2x SSPE (20).…”

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Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker in Saccharomyces cerevisiae.

Miyajima¹,

Miyajima²,

Arai³

et al. 1984

Mol. Cell. Biol.

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The R388 plasmid-encoded drug-resistant type II dihydrofolate reductase gene (R * dhfr) was expressed in Saccharomyces cerevisiae by fusing the R * dhfr coding sequence to the yeast TRP5 promoter. Yeast cells harboring these recombinant plasmids grew in media with 10 ,ug of methotrexate per ml and 5 mg of sulfanilamide per ml, a condition which inhibits the growth of wild-type cells. Addition of a 390-base-pair fragment from the 3'-noncoding region of TRP5 downstream from R * dhfr increased expression. Presumably, the added segment promoted termination or polyadenylation or both of the R dhfr transcript. The activity of the plasmid-encoded dihydrofolate reductase and the copy number of the R dhfr plasmid in cells grown in drug-selective media were higher by one order of magnitude than those grown in nutritionselective media. Plasmid copy number, as well as the plasmid-encoded enzyme level, decreased when cells were selected for prototrophy. In drug-selective media, the plasmid-encoded enzyme level and the content of R * dhfr transcripts were nearly constant in cells harboring R * dhfr plasmids containing different yeast promoters. In contrast, the plasmid copy number and ,-lactamase activity encoded in cis by plasmids were much higher when R * dhfr was associated with the weak TRP5 promoter than when it was fused to the strong ADCJ promoter. These results indicate that plasmid copy number, i.e., gene dosage of R * dhfr, correlates inversely with the strength of the promoter associated with R * dhfr, and cells with a higher plasmid copy number were enriched in drug-selective media. The transformation efficiency of R * dhfr fused to the ADCJ promoter was almost the same on drug-selective plates as on nutrition-selective plates, indicating that R * dhfr is suitable as a dominant selective transformation marker in S. cerevisiae.

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Isolation and characterization of yeast mutants auxotrophic for 2′-deoxythymidine 5′-monophosphate

Cited by 68 publications

References 33 publications

High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces

High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces

Cloning, purification, and properties of Candida albicans thymidylate synthase

Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker in Saccharomyces cerevisiae.

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