Figure 6
Construction and analysis of cdc27-Q1 mutant yeast. A. Upper part: Schematic of the cdc27+ gene region (3.1 kb HindIII-BamHI region) showing location of oligonucleotides used for PCR amplification. (Key to oligonucleotides: Aw = CDC27-Q1W-DIAG2, Am = CDC27-Q1M-DIAG2, B = CDC27-B, C = CDC27-SEQ2005, D = CDC27-H, X = CDC1-AB, and Y = CDC1-XY – see Material and methods for sequences). The boxes indicate approximate positions of cdc27+ exons. The NotI site shown is found in the cdc27-Q1 allele only. Lower part: Genomic DNA prepared from wild-type (WT) and cdc27-Q1 (Q1) strains was amplified using the primer pairs shown. The D+B PCR product from cdc27-Q1 alone can be digested with NotI (data not shown). The primer pair X and Y amplify an unrelated region of genome, and were included as a control. Molecular weight markers (kb) are shown to the right of the gel. B. Wild-type (cdc27+, left) and cdc27-Q1 (right) cells plated on YE medium and incubated for 3 days at 32°C. Mutation of the DPIM did not affect the efficiency of colony formation or growth rate. See text for details.