Figure 2

Pentapeptide scanning mutagenesis (PSM) strategy. A schematic representation of the mutagenesis and cloning strategies used to create the cdc1 PSM alleles. Plasmid pBR322-Cdc1 (top left) carries a cdc1⁺ cDNA on a BamHI-BamHI fragment. This plasmid was transformed into E.coli FH1046 containing pHT385. Following mating with E.coli DS941(see Methods), plasmids in which the Tn4430 transposon had inserted into the cdc1⁺ ORF (indicated as pBR322-Cdc1-Tn, top right) were identified by restriction mapping and the bulk of the Tn4430 sequences excised by KpnI digestion and self-ligation of the digested plasmids. The mutant alleles were then transferred into pREP3XH6BN, a fission yeast expression vector, and either pBTM116 or pBTM116BN, budding yeast two-hybrid bait vectors.