5 foa

Manufactured by US Biological

5-FOA is a chemical compound used in laboratory settings. It is a substrate for 5-fluoroorotic acid decarboxylase, an enzyme involved in pyrimidine biosynthesis. 5-FOA can be used as a selection agent in yeast genetic studies.

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Lab products found in correlation

Canavanine, by Merck Group (1 mentions) 5 fluoroorotic acid, by US Biological (1 mentions) Uracil, by Merck Group (1 mentions) Fluconazole, by LKT Laboratories (1 mentions) Nystatin, by Merck Group (1 mentions) Verapamil, by Merck Group (1 mentions) Fk506, by AG Scientific (1 mentions) Valinomycin, by AG Scientific (1 mentions) Cyclosporine a, by AG Scientific (1 mentions) Doxorubicin, by AG Scientific (1 mentions) Geneticin, by Santa Cruz Biotechnology (1 mentions) Hygromycin b, by Thermo Fisher Scientific (1 mentions) 5 fluoroorotic acid 5 foa, by US Biological (1 mentions)

8 protocols using 5 foa

Identifying Cells with Gross Chromosomal Rearrangements

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Cells with GCRs were identified by simultaneous inactivation of CAN1 and URA3 on chromosome V, indicated by resistance to canavanine and 5-FOA (Can^r 5-FOA^r). Cultures were grown for 2 days in ≥ 10 ml of YPD media. Viable cell counts were determined by plating dilutions on YPD agar plates, and cells with GCRs were identified by plating 0.25–15 ml on synthetic media lacking arginine and uracil and supplemented with 60 mg/liter canavanine (Sigma) and 1 g/liter 5-FOA (US Biological). The rate of accumulating GCRs was calculated as previously described (Schmidt et al. 2010b (link)).

Campos-Doerfler L., Syed S, & Schmidt K.H. (2017). Sgs1 Binding to Rad51 Stimulates Homology-Directed DNA Repair in Saccharomyces cerevisiae. Genetics, 208(1), 125-138.

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Functional Validation of Rab Mutants via Plasmid Shuffling

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Plasmid shuffling assays were performed to test whether Rab HVD mutants and chimeras are functional in vivo. ypt1Δ or ypt31Δypt32Δ null mutants were maintained by a copy of YPT1 or YPT31 on a centromeric URA3 plasmid. Shuffling strains were transformed with centromeric LEU2 plasmids containing Rab genes under their endogenous promoters. Transformed yeast were plated on -Leu synthetic dropout media to allow for cells with functional constructs to lose the URA3 maintenance plasmid. After 2–3 days of growth transformed cells were resuspended in yeast nitrogen base, diluted to an OD₆₀₀ of 0.5, then serially diluted onto -Leu and synthetic complete media with 3.9 mM 5-fluoroorotic acid (5-FOA; US Biological). Cells were grown at 30°C unless otherwise noted for 2–3 days prior to imaging.

Thomas L.L., van der Vegt S.A, & Fromme J.C. (2018). A steric gating mechanism dictates the substrate specificity of a Rab-GEF. Developmental cell, 48(1), 100-114.e9.

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Yeast Strain Characterization and Assays

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Yeast strains used in this study had an A364A background and their genotypes are listed in Supplemental Material, Table S1 in File S1. YPD liquid media was prepared containing 1% yeast extract, 2% peptone, and 2% dextrose, 0.01 mg/ml adenine. YPD solid media was prepared the same way as liquid media and contained 2% agar. Camptothecin (CPT) (Sigma [Sigma Chemical], St. Louis, MO) was made as a 10 mg/ml stock in dimethyl sulfoxide (DMSO) and added to a final concentration of 20 µg/ml in YPD solid or liquid media containing 25 mM pH 7.4 [4-(2-hydroxyethyl)-1-pipera-zineethanesulfonic acid (HEPES; Fisher Scientific, Fair Lawn, NJ)]. Next, 99% pure methyl methanesulfonate (MMS) (Sigma) was added to a final concentration of 0.01% in YPD solid media. MMS was diluted 1:10 in DMSO and added to YPD liquid media to a final concentration of 0.01%. Agar plates containing MMS were made within 2 days of use to prevent degradation. 5-FOA (US Biological Life Sciences, Salem, MA) was used at a final concentration of 1 g/liter in URA dropout plates supplemented with 50 mg/liter uracil (Sigma).

Bloom M.S., Koshland D, & Guacci V. (2017). Cohesin Function in Cohesion, Condensation, and DNA Repair Is Regulated by Wpl1p via a Common Mechanism in Saccharomyces cerevisiae. Genetics, 208(1), 111-124.

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Multidrug Screening in Yeast Model

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FK506, cyclosporine A, doxorubicin, and valinomycin were purchased from A.G. Scientific (San Diego, CA). Fluconazole was from LKT Laboratories (Saint Paul, MN). Verapamil, and nystatin were from Sigma Aldrich (Saint Louis, MO). 5-FOA and G418 were from US Biological (Swampscott, Massachusetts). E. coli lipids (Polar Extract) was purchased from Avanti (Alabaster, AL), n-dodecyl-β-D-maltopyranoside (DDM) was from Inalco (Italy).

Swartz D.J., Singh A., Sok N., Thomas J.N., Weber J, & Urbatsch I.L. (2020). Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions. Scientific Reports, 10, 3224.

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Yeast Strains and Cultivation Protocols

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Strains used in this study (Supplementary Table 6) are derivatives of the S288c strain RDKY5964^{20 (link)}, with exception of strain AH109 (Clontech Laboratories) that was used for Y2H analysis. Strains were cultivated at 30 °C in yeast extract-peptone-dextrose media (YPD) or appropriate dextrose-containing synthetic dropout (SD) medium for selection of plasmids markers, lacking lysine (Lys⁻) or threonine (Thr⁻) (to select for lys2-10A or hom3-10 frameshift revertants, respectively), or SD medium lacking arginine (Arg⁻) supplemented with 60 mg/L canavanine, to select canavanine-resistant (Can^R) mutants. 5-fluoroorotic acid (5-FOA, US Biological) plates were done in SD medium supplemented with 1 g/L 5-FOA. Antibiotics were used at the following final concentrations: 200 μg/mL geneticin (Santa Cruz Biotechnology), 300 μg/mL hygromycin B (Thermo Fisher Scientific), and 100 μg/mL nourseothricin (clonNAT, Werner BioAgents). Gene deletions and gene tagging were performed using standard PCR-based recombination methods^{51 (link),52 (link)}, followed by confirmation by PCR. Tags and junctions were confirmed by PCR and sequencing. Yeast strains carrying mutations in MLH1, MLH2, or PMS1 genes, were generated by pop-in/pop-out strategy using pRS306-based integrative vectors^{51 (link)}, and were confirmed by sequencing.

Reyes G.X., Zhao B., Schmidt T.T., Gries K., Kloor M, & Hombauer H. (2020). Identification of MLH2/hPMS1 dominant mutations that prevent DNA mismatch repair function. Communications Biology, 3, 751.

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Yeast Genetic Manipulation Techniques

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Table S1 lists the yeast strains used in this study. Yeast genetic methods including mating, sporulation, dissection, and transformations were conducted according to standard procedures⁴⁷. Yeast strains were grown at indicated temperatures in either YPD (2% peptone, 2% dextrose, 1% yeast extract) or selective minimal media lacking appropriate amino acids and supplemented with 2% dextrose and 5-fluoroorotic acid (5-FOA; United States Biological) as needed at 1.0 mg/mL. For growth analysis on plates, indicated strains were grown in YPD liquid media to mid-log phase (OD₆₀₀ ~0.5), serially diluted, and equal numbers of cells were spotted onto YPD media for growth at the indicated temperatures. For Y2H analysis, vectors expressing GBD and GAD fusions were transformed into the reporter strain (PJ69-4A) or nup42Δ reporter strain (SWY6432) and selected on –Trp –Leu synthetic media. The resulting strains were then grown to mid-log phase, serially diluted, and equal numbers of cells were spotted onto –Trp –Leu or –Trp –Leu –His –Ade media. The plates were then incubated at 23°C for the indicated number of days.

Adams R.L., Mason A.C., Glass L., A.d.i.t.i, & Wente S.R. (2017). Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells. Traffic (Copenhagen, Denmark), 18(12), 776-790.

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Yeast Strain Construction and Culture

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S1 Table lists the yeast strains used in this study. Yeast genetic methods including mating, sporulation, dissection, and transformations were conducted according to standard procedures. Yeast strains were grown at indicated temperatures in either YPD (2% peptone, 2% dextrose, 1% yeast extract) or selective minimal media lacking appropriate amino acids and supplemented with 2% dextrose and 5-fluoroorotic acid (5-FOA; United States Biological) as needed at 1.0 mg/mL. For split Venus tagging, haploid strains were transformed with either VN:HIS3MX or VC:KANMX6 integration cassettes. For haploid strains where no integrations were confirmed, diploids were transformed, integration was confirmed by PCR and western blot, and strains were sporulated and dissected. No viable tagged haploids were recovered from these diploids.

Adams R.L, & Wente S.R. (2020). Dbp5 associates with RNA-bound Mex67 and Nab2 and its localization at the nuclear pore complex is sufficient for mRNP export and cell viability. PLoS Genetics, 16(10), e1009033.

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Yeast Strains and Growth Conditions

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Saccharomyces cerevisiae strains were grown at 30 °C in YPD or SC media (Rose et al. 1990 ). The SC-His+Gal medium used to monitor the cryptic initiation phenotype contained 2% galactose. To assess defects in telomeric silencing, SC media contained 0.1% 5-fluoroorotic acid (5-FOA; USBiological, F5050). S. cerevisiae strains used in this study are listed in Table S1. KY strains are isogenic with FY2, a GAL2 + derivative of S288C (Winston et al. 1995) . KA strains were derived from crosses originating with strains from a library of histone H3 mutants (Dai et al. 2008) . Strains were made by standard methods for genetic crosses or gene replacements (Rose et al. 1990; Vidal et al. 1991) . Strains containing a mutation in RTF1 were identified during tetrad analysis by the presence of an HA-tag sequence, using PCR amplification. Strains containing the htb1-K123R allele were identified by restriction enzyme digestion as previously described (Tomson et al. 2011) . For phenotypes assessed by replica plating, strains were first purified on YPD. For serial dilution analysis, strains were grown overnight in liquid YPD, diluted to OD600 = 0.8, and 3 µl of five-fold serial dilutions were spotted onto SC-His+Gal or SC Complete media.

Shirra M.K., Kocik R.A., Ellison M.A., & Arndt K.M. (2020). Opposing functions of the Hda1 complex and histone H2B mono-ubiquitylation in regulating cryptic transcription initiation inSaccharomyces cerevisiae.

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