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. 2004 Dec;3(6):1533–1543. doi: 10.1128/EC.3.6.1533-1543.2004

FIG. 6.

FIG. 6.

Life without END. The cse4Δ::kanMX4 strain R332-5D (cse4::kan) carrying wild-type CSE4 on plasmid pRB163 was crossed with CSE4 wild-type strain R332-4C (WT) and cured of plasmid to obtain the heterozygous diploid strain R358. R358 was then transformed with PstI-linearized plasmid pRB588 to replace the cse4Δ::kanMX4 allele with GAL1-driven cse4Δ129::TRP1. A Kans transformant (R365) was picked and sporulated. Dissected tetrads each yielded 2 Trp+ spores and 2 Trp− spores, and all 22 Trp+ spores were also glucose sensitive. DNA from the indicated strains was cleaved with EcoRI, separated on an agarose gel, transferred to a nylon membrane, and hybridized with a probe homologous to DNA encoding the Cse4 END. The blot was stripped and rehybridized with a probe homologous to DNA encoding the Cse4 HFD. The HFD probe extends beyond the CSE4 coding region and thereby detects the cse4Δ::kan genomic EcoRI fragment.