Journal: Molecular Cell

Article Title: Cancer-Associated Gain-of-Function Mutations Activate a SWI/SNF-Family Regulatory Hub

doi: 10.1016/j.molcel.2020.09.024

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Article Snippet: 5-Fluoroorotic Acid (5FOA) , Toronto Research Chemicals , Cat#F59500.

Techniques: Virus, Recombinant, Purification, SYBR Green Assay, Sequencing, Plasmid Preparation, Software

Journal: PLoS Biology

Article Title: Unifying the mechanism of mitotic exit control in a spatiotemporal logical model

doi: 10.1371/journal.pbio.3000917

Figure Lengend Snippet: (A) The model correctly predicted 81% of the 140 tested literature phenotypes . (B) The model often failed at predicting the phenotype of cells with a genotype that mixes overexpression with other mutations, such as the rescue of the temperature-sensitive alleles cdc15-2 and dbf2-2 by overexpression of CDC5 . (C) The model predicts that overexpression of CDC5 cannot rescue the mob1Δ mutation. (D) Spot test confirming the model prediction that overexpression of CDC5 cannot rescue full deletion of MOB1 . A mob1Δ strain kept alive by provision of a CEN-MOB1 plasmid with uracil selection was transformed with either a 2 − μ m plasmid bearing MOB1 or CDC5 or an empty plasmid. The CEN-MOB1 plasmid was counterselected by addition of 5FOA, showing that moderate overexpression of CDC5 is not sufficient for rescue of the mob1Δ phenotype. (E) The localisation state of MEN proteins on the SPBs in the 3 physiological stages of mitotic exit in the model. Steady states determined from synchronous update scheme. (F) Comparison of (a)symmetry of SPBs in the steady states of wild-type and bfa1Δ cells. All simulation data can be found in . CDK, Cyclin-Dependent Kinase; MEN, Mitotic Exit Network; SC-LEU, Synthetic Complete media lacking leucine.; SPB, Spindle Pole Body.

Article Snippet: Counterselection of plasmids with uracil selection was achieved by addition of 750- μ g/ml 5FOA (FluoroChem, United Kingdom).

Techniques: Over Expression, Mutagenesis, Spot Test, Plasmid Preparation, Selection, Transformation Assay, Comparison

Journal: eLife

Article Title: Specialization of the chromatin remodeler RSC to mobilize partially-unwrapped nucleosomes

doi: 10.7554/eLife.58130

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Article Snippet: Chemical compound, drug , 5-Fluoroorotic Acid (5FOA) , Toronto Research Chemicals , Cat# F59500 , .

Techniques: Recombinant, Purification, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro

doi: 10.1073/pnas.2001451117

Figure Lengend Snippet: PCR amplification of URA5 in representative 5FOA-resistant mutants showing T1, TCN12 (full-length), and TCN12 solo long terminal repeat (LTR) insertions. (A) Amplification from genomic DNA of mutants recovered from the lungs of mouse 3. (B) Amplification from genomic DNA of mutants isolated after growth at 30° or 37° in vitro. (C) Illustration of the T1 DNA transposon with 11-bp terminal inverted repeats and the TCN12 retrotransposon with the gag-pol-polyprotein and 150-bp LTR direct repeats. The integrase (INT) and reverse transcriptase (RT) open reading frames are indicated. Amplification of a wild-type (WT)-sized URA5 band in addition to the larger band diagnostic of TE insertion presumably reflects loss of the TE in a subpopulation of cells during nonselective growth prior to isolation of genomic DNA.

Article Snippet: Mutants resistant to 5FOA were selected on synthetic complete medium (SC; 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 0.13% Hartwell’s complete amino acid mix, and 2% agar) supplemented with 1.3 μg/mL uracil and 1 μg/mL 5FOA (US Biological).

Techniques: Amplification, Isolation, In Vitro, Reverse Transcription, Diagnostic Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro

doi: 10.1073/pnas.2001451117

Figure Lengend Snippet: TE insertions (T1 and TCN12) in URA3 and URA5 of 5FOA-resistant mutants recovered from mice (M1–M8; color coded by organ) and inoculum cultures (C1–C7; green font). C7 was used to infect both M7 and M8. Highlighted in gray are TE insertions from mouse 3 and culture 3. The organ from which each mutant was recovered is indicated: lungs (L, blue), kidneys (K, red), brain (B, purple). Arrows represent the forward or reverse integration of each TE. Uppercase letters are exon sequences, and lowercase letters are intron sequences. Underlined sequences indicate those sequences that are duplicated following TE integration. For some of the TCN12 insertions, only a solo LTR was detected.

Article Snippet: Mutants resistant to 5FOA were selected on synthetic complete medium (SC; 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 0.13% Hartwell’s complete amino acid mix, and 2% agar) supplemented with 1.3 μg/mL uracil and 1 μg/mL 5FOA (US Biological).

Techniques: Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro

doi: 10.1073/pnas.2001451117

Figure Lengend Snippet: Temperature-dependent mutagenesis is driven by TE insertions. (A) Drug-resistance rates for 5FOA, rap/FK506, and 5FC when growth before drug selection was at either 30° (blue) or 37° (red) in vitro. (B) Insertion rates of individual TEs into URA3 or URA5 (URA3/5). (C) Distribution of T1 and TCN12 insertions in URA3, URA5, and FRR1 in drug-resistant mutants isolated from 37° cultures; the distribution at each locus is significantly different (P < 0.001 by χ2 contingency test). (D) Insertion rates of T1 and TCN12 into the FRR1 gene. Error bars are 95% confidence intervals (CIs); rates are statistically different if error bars do not overlap. Asterisks indicate no insertions were detected; the rate shown was calculated assuming one event.

Article Snippet: Mutants resistant to 5FOA were selected on synthetic complete medium (SC; 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 0.13% Hartwell’s complete amino acid mix, and 2% agar) supplemented with 1.3 μg/mL uracil and 1 μg/mL 5FOA (US Biological).

Techniques: Mutagenesis, Selection, In Vitro, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro

doi: 10.1073/pnas.2001451117

Figure Lengend Snippet: The 5FOA resistance rates and TE insertion rates in clinical and environmental isolates. (A) 5FOA resistance rates for clinical strains NIH12, 528, AD7-71, and environmental strain NIH433 grown at 30° (blue) or 37° (red). The ratios of the rates at the two temperatures are indicated by the brackets. (B) Insertion rates of TEs into URA3/5 in clinical isolates NIH12 and 528. Error bars are 95% CIs; rates are statistically different if error bars do not overlap. Asterisks indicate no insertions were detected; the rate shown was calculated assuming one event.

Article Snippet: Mutants resistant to 5FOA were selected on synthetic complete medium (SC; 0.17% yeast nitrogen base, 0.5% ammonium sulfate, 0.13% Hartwell’s complete amino acid mix, and 2% agar) supplemented with 1.3 μg/mL uracil and 1 μg/mL 5FOA (US Biological).

Techniques: