Hi there,
The strain has CEN.PK2-1C background ( MATa ura3-52 trp1-289 leu2-3,112 his3Δ) . I have checked the strain phenotype towards tryptophan: it is unable to grow without tryptophan and becomes trp+ when transformed with TRP1 containing plasmid. However, the strain is unable to grow efficiently on 5FAA plates (MC supplemented with 0.5g/L 5FAA from Sigma). I followed the described protocol by the book: first dissolving 5FFA powder in EtOH to get a 10% solution (100mg/mL) and using it as a x200 stock solution. The plates seem OK as they are able to counterselect TRP1 plasmid in other yeast background (W303). What do you reckon? Thanks for the help.
Hi Dominique, it's a perplexing problem. Since trp1-289 is a point mutation and not a deletion I wonder whether it is leaky or whether your strain picked up a partial suppressor? You do have that allele in a different genetic background that you can test?
Michael J. Benedik : thanks for your reply. I'm afraid this strain is the only I have in hands bearing this allele... Could it be the trp1-289 mutant retains enough catalytic activity with 5FAA derivative to make it toxic but unsufficient activity on TRP1 natural substrate to exhibit a trp+ phenotype?
That what exactly what I was thinking, but I just did a quick check on the genotype and the allele is a nonsense codon at 403 out of 675nts. It is revertible.
You might play around with FAA concentration, drop it a little and see if the toxicity goes down.
Thanks for the info. I was also thinking about the possibility of lowering 5FAA concentration but 0.5g/L is already in the lower range... I'll see... After the summer break! Cheers.
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