In article <199507111439.HAA13763 at net.bio.net>, U09668 at UICVM.CC.UIC.EDU (E
> Dear fellow yeasters:
> I have a strain with a genomic disruption of a gene using a URA3 marker, and
> would like to directly transplace the disrupted gene with an intact copy of
> gene. I was thinking about directly selecting the transformants on FOA
> medium, and was wondering if anyone out there has ever tried this. I don't
> have a selectable marker for the gene I would like to introduce, but I can
> select real transformants as opposed to URA3 revertants, by phenotype.
> Does this approach seem reasonable, and can anyone give me any possible
> or hints about this strategy? Please e-mail me directly with any advice.
> you very much.
>>> E Li
> Univ. of Ill. at Chicago, Dept. of Biological Sciences
(This posting is for others that may also be interested.)
>URA3 expression cannot be directly selected against because of its fairly
>long phenotypic halflife ("phenotypic lag"), and because FOA selection in a
>URA3(+) strain is toxic, as opposed to growth static. Direct selection on
>FOA will only identify those cells that were URA3(-) before the
I cannot agree here with Steven. I use 5-FOA to screen for recombination
between 2 overlapping YACs, one of which contains URA3. recombination
results in URA3 deletion and growing cells throw URA3 containing fragment
away. I use proline based medium for 5-FOA selection.
>The best solution is to cotransform the gene fragment with a small amount
>of a closed circular plasmid with a different selectable marker (eg: TRP1
>or LEU2). Select for the cotransformed plasmid marker. Then either
>replica print to FOA or check the phenotype of the transformants directly,
>if you can. I typically get about 0.1% to 1% of the original transformants
>that show a gene conversion that removes the URA3 marker.
>>Steven A. Haney
>Broach Lab/Dept. of Mol. Biol.
>shaney at watson.princeton.edu