I am working on a project which involves selecting alleles conferring
partial resistance to FOA in Saccharomyces. I see from the literature
that 1 g/l FOA is recommended for selecting for the loss of URA3
plasmids, from which I conclude that null mutants at this locus grow
fine at this concentration. I gather that ura5 mutants are also
resistant to this drug concentration. However, I would like to find
less efficient resistance mechanisms.
So my question is:
Can anybody warn me _away from_ major pitfalls such as known and not
interesting genotypes conferring resistance to, say, 0.1 g/l or even
0.01 g/l, but not to 1 g/l?
Preliminary experiments indicate that my URA3 URA5 strain does not
grow at even 0.01 g/l. However, given the price of the drug, I have a
hard time believing that people routinely use a 100X excess of the
stuff. My guess is that there are known alternate resistance
mechanisms that justify the use of 1 g/l to select the ura3 genotype.
If there appears to be interest, I will post a summary of responses
received both on the net and by email. If you would like to keep your
ideas just between the two of us, reply by email mentioning that fact
and I will of course respect your wish. (I only say this because I
was once slightly nonplussed when somebody simply bounced my emailed
response to his or her query out to the whole net. Information should
circulate as freely as possible in this medium, but it's just reality
that we may say things a little differently one-on-one vs at large
and we all have the odd secret)
Thanks for your time.
Apologies to makers of FOA if I am wrong that the 1 g/l concentration
really is necessary for routine counterselection of the URA3 marker!
John Bratty bratty at medcn.umontreal.ca
Dept. de Biochimie
Universite de Montreal (514) 343-6111, ext. 5165
Montreal, Canada H3C 3J7