Figure 3.

Loss of silencing and slow growth recreated by an AUG to AUU change in the CBF5 initiation codon. (A) The expression of the tRNA^SUP4 gene and the HIS3 gene on media containing glucose (represses pol II promoter) or galactose (induces pol II promoter). The tRNA^SUP4 gene is always expressed when present, tested by the suppression of a chromosomal ade2-101 mutation. In the original mutant (art1-1, which is derived from strain YM2062), expression of HIS3 from the GAL1 promoter requires galactose induction. To show that this galactose-inducible His⁺ phenotype is due solely to the CBF5 AUG to AUU mutation, either the mutated or wild-type CBF5 gene was introduced into an unrelated strain (W3031A) on a plasmid, and the chromosomal CBF5 was deleted. The AUU mutation conferred galactose-inducible HIS3 expression. (B) Recreation of the AUU translational initiation codon in CBF5 (W3031aΔcbf5/pSUP4^oCBF5-AUU) also recreates the slow growth phenotype of the original mutant compared with the same construct with the wild-type AUG in the plasmid-borne CBF5 gene.