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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Biochemistry. 2010 Jun 1;49(21):4440–4449. doi: 10.1021/bi100394j

FIGURE 2.

FIGURE 2

Experimental strategy designed to determine whether a suppressor is due to a second-site mutation in pdr5S558Y. A, B Suppressors were isolated as chromosomal mutations in JG2011 which has two PDR5 cassettes. The first has the coding sequence replaced with KANMX4 but retains the upstream and downstream flanking regions. This is separated by plasmid sequences including the selectable marker URA3 from a second copy of PDR5 containing S558Y. C, In this illustration, it is assumed that the new mutation (*) lies in S558Y rather than in a second gene (the case for all of the mutants recovered as S558Y suppressors). D, Loss of the plasmid sequences containing URA3 and one of two PDR5 cassettes (either pdr5::KANMX4 or pdr5S558Y ) occurs by homologous recombination. These events result in Ura- colonies that are selected on plates containing 1 mg/ml 5-FOA. These are tested on gen (200 mg/l) and clo-containing medium (7.5 μM). E, If the suppressor is attributable to a second mutation in the S558Y-bearing copy, clor gens, and clos genr recombinants will be recovered as shown.