YEPD rich medium [1% yeast extract (BD Bacto), 2% peptone (BD Bacto), 2% dextrose (J. T. Baker)] was used unless other specific medium is mentioned. For selection, YEPD was supplemented with 300 mg/L Hygromycin B (InvivoGen), 400 mg/L G418—Geneticin (Santa Cruz) or both. For selecting ΔURA3 strains, Synthetic Complete (SC) medium was prepared with 5-FOA [2% dextrose, 0.17% yeast nitrogen base (Difco), 0.14% amino acid dropout, 0.5% ammonium sulfate (Sigma), 0.08% 5-Fluoroorotic acid (US Biological) and the required amino acids]. SC medium lacking uracil was used for selecting cells with intact URA3. Solid media were prepared by addition of 2% agar (Difco) to any of the liquid media listed above. The strains used in this study are diallel strains (Shapira et al., 2014 (link)) and manipulated strains as described in the text that were made in two genetic backgrounds: S288c (Winzeler et al., 1998 (link)) and SK1(Kane and Roth, 1974 (link)).
5 fluoroorotic acid
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5-fluoroorotic acid is a chemical compound used in biological research and laboratory applications. It serves as a precursor or intermediate in various biochemical and molecular biology experiments. The core function of 5-fluoroorotic acid is to facilitate specific research processes, but a detailed description of its intended use is not provided to maintain an unbiased and factual approach.
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7 protocols using 5 fluoroorotic acid
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Yeast Growth Media Preparation
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Functional Validation of Rab Mutants via Plasmid Shuffling
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Quantifying Gross Chromosomal Rearrangements
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Rates of gross chromosomal rearrangements (GCRs) were determined using a standard protocol (Putnam and Kolodner, 2010) (link). Briefly, pre-cultures of S. cerevisiae cells harboring a CAN1::URA3 reporter on chromosome 5 were grown in SC-Ura medium and plated out on YPD plates to obtain colonies that formed from single cells. Eight colonies were excised from the plates for each condition and used to inoculate larger cultures in YPD (control strains: 50 ml; strains with stabilized interaction: 2 ml), which were grown to stationary phase at 30 °C. The number of viable cells was determined by plating a serial dilution (10 -6 ) on nonselective YPD plates. The total number of GCR events was determined by plating the remaining culture on SC-Arg plates that were supplemented with 50 mg/L L-canavanine (Sigma C9758) and 1 g/L 5'-fluoroorotic acid (US Biological Life Sciences F5050) to select against both CAN1 and URA3. No more than 10 9 cells were spread on each selection plate and the plates were incubated at 30 °C for two days (YPD) and three to five days (selection).
Afterwards, the clones were counted and GCR rates as well as confidence intervals were calculated by fluctuation analysis using the maximum likelihood method in the web tool FALCOR (B. M. Hall et al., 2009) (link) that was kindly made accessible by the Liang lab under https://lianglab.brocku.ca/FALCOR/.
Afterwards, the clones were counted and GCR rates as well as confidence intervals were calculated by fluctuation analysis using the maximum likelihood method in the web tool FALCOR (B. M. Hall et al., 2009) (link) that was kindly made accessible by the Liang lab under https://lianglab.brocku.ca/FALCOR/.
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Measuring Gross Chromosomal Rearrangements
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Measuring Gross Chromosomal Rearrangements in Yeast
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Rates of gross chromosomal rearrangements (GCRs) were determined using a standard protocol85 (link). Briefly, pre-cultures of S. cerevisiae cells harboring a CAN1::URA3 reporter on chromosome 5 were grown in SC-Ura medium and plated out on YPD plates to obtain colonies that formed from single cells. Eight colonies were excised from the plates for each condition and used to inoculate larger cultures in YPD (control strains: 50 ml; strains with stabilized interaction: 2 ml), which were grown to stationary phase at 30 °C. The number of viable cells was determined by plating a serial dilution (10−6) on non-selective YPD plates. The total number of GCR events was determined by plating the remaining culture on SC-Arg plates that were supplemented with 50 mg/L L-canavanine (Sigma C9758) and 1 g/L 5′-fluoroorotic acid (US Biological Life Sciences F5050) to select against both CAN1 and URA3. No more than 109 cells were spread on each selection plate and the plates were incubated at 30 °C for 2 days (YPD) and 3–5 days (selection). Afterwards, the clones were counted and GCR rates as well as 95% confidence intervals were calculated by fluctuation analysis using the maximum likelihood method in the web tool FALCOR109 (link) that was kindly made accessible by the Liang lab under https://lianglab.brocku.ca/FALCOR/ (no version data available, as used in September 2020).
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Yeast Strains and Genetic Manipulations
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The yeast strains and plasmids used in this study are listed in Supplemental Material, Table S1 and Table S2 . Yeast was grown and manipulated according to standard procedures (Sherman 1991 (link)). Yeast was grown on full medium (YPD) and selective minimal plates (YM), and plates containing 5-fluoro-orotic acid (US Biological) were used to select against URA3 . Chromosomal integration of sir4 alleles was obtained by transferring them onto a yeast integrating plasmid (pRS306, URA3 -marked), and introducing them into yeast strains by integrative transformation followed by loop-out on 5-FOA medium. Semiquantitative mating assays were performed by generating serial dilutions (1:10, start OD600 of one) of the respective strain in a microtiter dish. For the growth control, cells were transferred to agar plates using a replica tool. An equal volume of the mating tester strain (suspension of 10 OD600 per milliliter) was then added to the strain in the microtiter well, and a replica of this mixture was transferred to a plate selective for the growth of diploids. Plates were incubated for 2–3 d at 30°.
pGBD-C2-sir4 plasmids were generated by excising a ClaI/BlpI fragment of sir4 alleles from pAE2289 or pAE2029, and inserting it into ClaI/BlpI-cleaved pAE1355. pGAD-C2 and pGBD-C2 plasmids encoding the coiled-coil domain of Sir4 were generated by inserting Sir4 fragments using ClaI and BglII.
pGBD-C2-
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Quantitative Measurement of Genome Instability
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A minimum of three independent spore clones were isolated from each mutant progeny pool arising from the SGA protocol and then grown as patches on a YPD-agar plate at 30 °C for two days and replica-plated onto CSM -Arg media containing 60 mg l−1 canavanine (Sigma) and 1 g l−1 5-fluoroorotic acid (US Biological). The number of papillae growing on the GCR medium was scored using a semi-quantitative scoring system as follows: 0, no papillae; 1, 1–5 papillae (this was on average the number of papillae observed with the leu2Δ control strain for the dGCR assay); 2, 6–15 papillae; 3, 16—a countable number of papillae (∼150–200); 4, papillae that were too many or too close together to count; 5, a lawn of papillae covering the entire patch (Fig. 1d ). Then the scores for all independent patches analysed for each mutant were averaged to generate a GCR strain score (Supplementary Data 1 ). Negative scores were assigned to strains that did not grow so that these strains could be ignored during the analysis.
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