Chapter 16 Isolation and Characterization of Mutants of Saccharomyces cerevisiae Able to Grow after Inhibition of dTMP Synthesis

Brendel, Martin; Fäth, Wolfgang W.; Laskowski, Wolfgang

doi:10.1016/s0091-679x(08)60329-5

Cited by 22 publications

(11 citation statements)

References 16 publications

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“…This concept was then adapted for use in yeast (Miyajima et al ., ). As the authors documented, methotrexate is inefficient at killing yeast cells unless sulphanilamide is added to block the de novo synthesis of dihydrofolate (Brendel et al ., ; Wickner, ). Similarly, we found that growth is completely inhibited in yeast grown in the presence of 20 n m methotrexate and 5 mg/ml sulphanilamide, but a limited level of growth is detected at all concentrations of methotrexate tested in cultures lacking sulphanilamide (Figure A).…”

Section: Resultsmentioning

confidence: 99%

Puromycin‐ and methotrexate‐resistance cassettes and optimized Cre‐recombinase expression plasmids for use in yeast

MacDonald

Piper

2015

Yeast

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Here we expand the set of tools for genetically manipulating Saccharomyces cerevisiae. We show that puromycin-resistance can be achieved in yeast through expression of a bacterial puromycin-resistance gene optimized to the yeast codon bias, which in turn serves as an easy to use dominant genetic marker suitable for gene disruption. We have constructed a similar DNA cassette expressing yeast codon-optimized mutant human dihydrofolate reductase (DHFR) that confers resistance to methotrexate and can also be used as a dominant selectable marker. Both of these drug-resistant marker cassettes are flanked by loxP sites allowing for their excision from the genome following expression of cre-recombinase. Finally, we have created a series of plasmids for low-level constitutive expression of cre-recombinase in yeast that allows for efficient excision of loxP-flanked markers.

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Section: Resultsmentioning

confidence: 99%

Puromycin‐ and methotrexate‐resistance cassettes and optimized Cre‐recombinase expression plasmids for use in yeast

MacDonald

Piper

2015

Yeast

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“…Similarly, in YPD-2 medium, growth of S. cerevisiae was not affected by 80 ,ug of methotrexate per ml, whereas it was completely inhibited by 40 ,ug of methotrexate per ml in the presence of 5 mg of sulfanilamide per ml (data not shown). Since tetrahydrofolate is required for the synthesis of adenine, glutamic acid, glycine, and methionine, complete removal of these components from the drug medium may cause severe growth inhibition (9,32).…”

Section: Resultsmentioning

confidence: 99%

“…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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“…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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“…The loss of mitochondrial DNA and the formation of respiration-deficient strains in S. cerevisiae grown in the presence of antifolate drugs (5,6,22,23) is due to the limitation of tetrahydrofolate formation and, hence, of 5,10-methylene tetrahydrofolate and l1-formyl tetrahydrofolate needed for the biosynthesis of both DNA and protein.…”

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Influence of the nuclear gene tmp3 on the loss of mitochondrial genes in Saccharomyces cerevisiae.

Zelikson¹,

Luzzati²

1982

Mol. Cell. Biol.

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The Saccharomyces cerevisiae tmp3 mutant is deficient in the mitochondrial enzyme complex that participates in the formation of one-carbon-group-tetrahydrofolate coenzymes, serine transhydroxymethylase, dihydrofolate reductase, and thymidylate synthetase, thus leading to multiple nutritional requirements of dTMP, adenine, histidine, and methionine. The tmp3 mutant quickly loses its mitochondrial genome even when grown on fully supplemented medium or on a high concentration of 5-formyl tetrahydrofolate, which replaces all the four requirements. A study of the loss of the mitochondrial genome by following several mitochondrial genetic markers showed that there was a preferential specific loss of a large region of the mitochondrial genome, covering mit ts983, E, Cr, and mit ts982 up to Or, and retention of the region of pr and mit tscs1297. A kinetic study showed that there was a preferentially rapid loss of the region covering the mit+ alleles ts983 to tscs9O2 at the rate of 10%o per generation.Inhibition of DNA synthesis in Saccharomyces cerevisiae cells, either by limitation of the precursor supply (4) or by deficiencies in other functions needed for its synthesis, such as cdc8 (19), results in the formation of respiratorydeficient cells.The loss of mitochondrial DNA and the formation of respiration-deficient strains in S. cerevisiae grown in the presence of antifolate drugs (5, 6, 22, 23) is due to the limitation of tetrahydrofolate formation and, hence, of 5,10-methylene tetrahydrofolate and l1-formyl tetrahydrofolate needed for the biosynthesis of both DNA and protein.A similar formation of respiratory-deficient cells by mutations leading to a limitation in the formation of thymidylate was described in mutants bearing tmpl allelic to cdc21 (4, 19) in fol mutants (15), and in tmp3 mutants (16).There are two types of mutants which are deficient in dTMP formation. The first one, tmpl, is deficient in thymidylate synthetase (2,12

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Chapter 16 Isolation and Characterization of Mutants of Saccharomyces cerevisiae Able to Grow after Inhibition of dTMP Synthesis

Cited by 22 publications

References 16 publications

Puromycin‐ and methotrexate‐resistance cassettes and optimized Cre‐recombinase expression plasmids for use in yeast

Puromycin‐ and methotrexate‐resistance cassettes and optimized Cre‐recombinase expression plasmids for use in yeast

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

Influence of the nuclear gene tmp3 on the loss of mitochondrial genes in Saccharomyces cerevisiae.

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