To generate SIR2OE strains, a genomic clone of SIR2 along with ∼500 bp of upstream sequence and ∼250 bp of downstream sequence was subcloned by PCR amplification with SIR2ecoriF and SIR2ecoriR primers, digested with EcoRI, and ligated into pRS306 cut with EcoRI to create the integration plasmid pRS306-SIR2. The plasmid was cut with BglII and transformed into UCC5179 and UCC5181 to generate strains UCC8908 and UCC8909. Correct integration was confirmed by PCR. These haploids were mated together to generate the diploid strain UCC8910. The SIR2hemi diploid was generated by transforming UCC8832 with pRS314-SIR2, mating to UCC5179, and subsequently curing the diploid of the plasmid.
