Figure 4.

(A) Generation of the GAL-RSP5 strain. Nucleotides 1–1,077 of the RSP5 gene (rsp5-Δ) were cloned into the pYES2 vector (Invitrogen) lacking a 2-μm replication origin. This was used to transform the FY56 yeast strain (ura3-52), selecting for growth on 2% galactose plates in the absence of uracil. The predicted recombination product, which was confirmed by PCR analysis of genomic DNA, is shown. (B) The RSP5 and GAL-RSP5 strains were grown at 30°C in minimal medium with 2% galactose to an OD₆₀₀ of 0.5. Aliquots of the culture were removed to either fresh galactose- or dextrose-containing medium and incubated for an additional 36 h. Total cell extracts were analyzed by SDS/PAGE and immunoblotting with antibodies against Rpb1, Rsp5, and the 70-kDa subunit of yeast replication protein A (RPA). Extracts from the RSP5 strain, grown in galactose (Gal.) and dextrose (Dex.), respectively, were analyzed in lanes 1 and 2, and extracts from the GAL-RSP5 strain were analyzed in lanes 3 and 4. Rsp5 protein from the GAL-RSP5 strain migrates slightly slower due to a 12-amino acid epitope at its amino terminus.