Figure 1.
Isolation of Bfa1 mutants that fail to induce mitotic arrest. (A) Schematic diagram of full-length BFA1 and BFA1-D8. The arrows represent two regions of imperfect direct repeats that are highly conserved among fungi: direct repeat 1 (DR1, residues 344-366) and DR2 (residues 409-422). Sequence alignment of DR1 and DR2 among fungi is shown. Identical amino acids are highlighted in black, and amino acids with high similarity are highlighted in gray. (B) Mutated Bfa1 residues in the screened mutants. Mutated bases by random mutagenesis with hydroxylamine are shaded. (C and D) Bfa1-D8M413I and Bfa1-D8G411E mutants failed to induce mitotic arrest. Δbfa1 (YSK8) cells carrying the indicated plasmids were arrested by α-factor and released into medium containing (C) galactose for overexpression or (D) 15 μg/ml nocodazole. At each time point after the release, (C) DNA content was analyzed by fluorescence-activated cell sorting (FACS; n = 50,000) and (D) cells with more than one bud were counted (n = 300). (D) The average of three independent counts is shown with SDs.