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Forsburg Lab Recipes: Fission Yeast Drugs and DNA damaging agents

This page provides information on drugs and supplements for a variety of purposes, including counterselection agents 5-FOA, canavanine, and FUDR, selection agents including G418, the spindle poison thiabendazole, and The Forsburg Lab Guide to DNA damaging agents.Please let us know if you have corrections, additions, or more information to include here!

Counterselection/selection Agents
These agents can be used to select for or against the indicated marker on a plasmid or in the genome
EMM + 5' FOA
Selects against: ura4+
5' FOA generates a toxic metabolite in Ura+ strains. (0.1% 5FOA, 2% dextrose, supplemented with low ura, ade, leu, his) NOTE: YNB contains thiamine, so these plates cannot be used for nmt induction. FOR ONE SLEEVE OF NORMAL 5FOA:
Have available sterile 2mg/ml uracil (this will not crash), 7.5 mg/ml stock solutions of ade, leu, and his. Use 3.75 mg/ml uracil.
  1. Autoclave 15 minutes: 10g agar in 250 ml MQ H2O, with stir bar, in 1 liter bottle.
    Weigh bottle before autoclaving
    Correct for loss of H2O by adding sterile MQ H2O.
    Store at 50*C until step 2 is completed.
  2. (The liquid volume should add up to 250 ml total for this step). To 198.3 ml MQ H2O, add:
    • 3.5 g Yeast Nitrogen Base with NH4 and without amino acids (Difco 0919-15-3)
    • 10 g dextrose
    • 0.5 g 5FOA (American Biorganics, Inc 07-00850; Toronto Research Chemicals cat. no. F595000)
    • 6.7 ml 3.75 mg/ml uracil
    • 15 ml 7.5 mg/ml ade
    • 15 ml 7.5 mg/ml leu
    • 15 ml 7.5 mg/ml his
    leave at 50*C, with occassional mixing until 5FOA is dissolved. Filter sterilize.
  3. YNB/amino acids/5FOA to sterile agar and mix carefully.
  4. Pour plates. Store at 4* in the dark. Note that standard cerevisiae media calls for anything from 0.1% to 1% FOA: for example, see this discussion from the bionet.yeast site. Your mileage may vary!
FUDR
Selects against: adh-tk+
Viral thymidine kinase makes the cells sensitive to this halogenated thymidine derivative.
Stock 5' fluoro-2'-deoxyuridine (Sigma F-0503) Add 10mg per 500ml agar.
Reference Genetics. 2000 154:599-607.
Canavanine
Selects against: CAN1 (from S. cerevisiae) in a can1- strain.
The can1+ gene encodes an arginine permease. The permease allows uptake of arginine. Thus, in the absence of the can1+ product, cells will be unable to import arginine, so the can1-1 strain must be Arg+. There also seems to be an effect on other positively charged amino acids--that is loss of the permease may affect uptake of histidine and lysine. Thus, for most utility, can1-1 strains should be His+ and Lys+. The can1 mutation in S. pombe renders cells resistant to the drug, but it has not yet been determined which gene is affected.
Stock 60 mg/ml (this is 1000X), dissolved in H2O and filter sterilized. Store at 4*C. Use at 60µg/ml.
Reference: NAR 19:1150 (1991)
G418
Selects for: kanMX cassette (G418 marker)
>Only works on YES plates--selection does not work in EMM. Use at 100-200 mg per liter. Store plates at 4*C in the dark. Can use minimal PMG medium (see below) > This is a difficult marker that can show substantial variations in expression, especially when integrated. Cells must be allowed phenotypic expression before selection; we plate on YES (non-seletive), then replica plate in a day to YES + G418. Use care in identifying true G418-resistant colonies. It is helpful to use a wild type (negative) and known kanR strain (positive) as controls every time. > Recent conversation (2/2008) on the pombelist mailing list suggest that PMG medium, which replaces the ammonium chloride in EMM with glutamate, will work fine with G418. Added bonus: Ura- cells grow more similarly to Ura+ cells on PMG.


Forsburg lab Guide to DNA damaging agents
General Information:
  • Damaging agents cause DNA breaks and are therefore toxic and carcinogenic. Use caution in handling these reagents: wear gloves and a lab coat, pour plates in the hood and dispose of waste in the chemical containers in the hood.
  • Use plates immediately (by the next morning) and/or store at 4*C in the dark once dry. Plates still retain damage-inducing ability for a few weeks if stored this way.
Bleomycin
effect Supposed to be a mimetic of gamma irradiation (double-strand breaks)
Liquid: 2.5 - 5mU/ml
S. pombe references: Mol Gen Genet 1997 254:389-99 MBC 11:1-11 (2002), MCB 21:1499-1508 (2001)
BrdU
effect easily visualized thymidine analogue. Requires thymidine kinase and thymidine uptake mechanism (strains typically carry HSV-tk+ and hENT; S. pombe cells can't take up BrdU on their own). Eventually leads to DNA damage.
Stock solution 10 mg/ml in water. Use at 1:50 in liquid cultures Wrap flasks in aluminum foil to protect from light.
Camptothecin (CPT)

effect Inhibitor of topoisomerase I; causes strand breaks in S phase
Plates:1uM to 20 uM
S. pombe references: Yeast 15:821-828 (1992); ; Mol Biol Cell. 2006 Jul;17(7):2976-85.
Hydroxyurea (HU)
effect Causes a range of lesions. Most often used to deplete the supply of nucleotides, leading to an S-phase arrest. Note that pombe is sensitive to HU at much lower levels than cerevisiae!
Plates: 5mM to 10mM. Wild type will eventually form colonies; sensitive strains will not.
Liquid: 12-15mM to arrest; e.g, for block and release. Reversable.
10mM will delay S phase.
For permanent arrest (irreversable), use 20-25mM
Stock: 1.5M, good for several days if stored at 4* in the dark.
S. pombe references: Genes Dev. 6:2035-2046 (1992)
Methyl Methanesulfonate (MMS)

effect Alkylating agent (causes a range of DNA damaged structures)
Plates 0.0025% to 0.0050%
Liquid: 0.007%
MMS plates should be kept for only a few days. Also, the fresher the MMS stock the better: apparently it becomes generally cytotoxic over time, but paradoxically loses some of its genotoxic effects. The nature of this chemical change is not clear. The same is true of EMS and may explain why some people consider EMS to be an inefficient mutagen in pombe. It's probably just fine as long as its very fresh, before other compounds develop. S. pombe references: MCB 22:4477-4490 (2002), MCB 22:3537-3548 (2002)
4-Nitroquinoline N-Oxide (4-NQO)

effect Supposed to be a UV mimetic; induces primarily G to A transitions
References: NAR 20:1283-1287 1992
Ultraviolet light (UV)

effect Causes a range of mutations.
Plates expose to 50-300 J/m2. Wrap plates in aluminum foil to prevent photoreactivation repair (may not be an issue in pombe)