FIG. 4.
Mutational analysis of Cef1p. Amino acids predicted to be critical for maintaining the structure of the Myb repeats of Cef1p were replaced with the indicated amino acid(s) as described in Materials and Methods. Substitution mutations were constructed in pCEF1, which is pRS415 (9) harboring a 2.5-kb genomic DNA fragment encompassing the CEF1 coding region. In addition to performing site-directed mutagenesis of the Cef1p gene, various domains of Cef1p were removed. pGAL1-CEF12R, Cef1p R1R2; pGAL1-CEF1CT, Cef1p CT including MLR3; pGAL1-CEF1dR1, Cef1p lacking R1; pGAL1-CEF1dR2, Cef1p lacking R2. These mutants were assayed for their abilities to rescue growth of the cef1-Δ1::HIS3 mutant by tetrad analysis and/or plasmid shuffle. Plasmid shuffle with the pRS425GAL1-based plasmids was performed on 5-fluoroorotic acid medium containing raffinose and galactose as the carbon sources. ++, rescue or growth comparable to that of wild-type CEF1; +, reduced rescue of growth and colony formation; +/−, poor rescue of growth; −, failure to rescue growth of the cef1-Δ1::HIS3 mutant or no growth; NA, not applicable.