To identify mutations that restore the activity of a defective ammonium transporter, natural suppressors or supprssors generated by mutagenesis can be identified.
Method Article
Multicopy suppressor screen
https://doi.org/10.1038/nprot.2007.124
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yeast
random mutagenesis
yeast complementation
To identify mutations that restore the activity of a defective ammonium transporter, natural suppressors or supprssors generated by mutagenesis can be identified.
Select suppressor mutants from the 31019b yeast strain(1) carrying the high copy number plasmid pDR-AtAMT1;1Thr460Ala by growing at 28ºC for 8 days on solid YNB medium supplemented with 3% glucose and 2 mM ammonium chloride.
Pick suppressors and amplify in liquid YNB medium supplemented with 3% glucose and 2 mM ammonium.
Isolate plasmid from yeast by CTAB extraction and use for E. coli transformation.
Amplify plasmids in E. coli, isolate and sequence.
When an inactive transporter Trans is subjected to a multi-copy suppressor screen, point mutations (single base changes, primarily) may occur spontaneously in the sequence of Trans. Such a spontaneous mutation occurs first in a single plasmid molecule, yielding a single Trans-Mut sequence. If Trans-Mut does not contribute to transport and growth, the cell will die and the sequence will be lost. If Trans-Mut does improve transport, it will initially function in (Trans)2(Trans-Mut)1 complexes, as Trans is present in vast excess. If activity is sufficient to lead to growth, Trans-Mut will replicate sufficiently to be observed by plasmid DNA sequencing. Reversion to the wild-type sequence in a single plasmid molecule would yield (Trans)2(wt)1 and (Trans)1(wt)2 complexes at the outset (wt3 would be a sufficiently small portion of the population that it would not contribute to growth). The absence of wild-type revertants from the suppressor screens suggests that these complexes are inactive.
(1) Marini, A. M., Soussi-Boudekou, S., Vissers, S. & André, B. A family of ammonium transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 4282-4293 (1997).
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