FIGURE 4.

Yeast growth phenotype for strains carrying mutations of the methionyl residues of the MMM triplet. Strain JL1Δ2Δ3u⁻ was transformed with plasmid pRS314 containing no gene (line 1), wild type ScANC2 gene (line 2), or scanc2 genes carrying mutations in the MMM cluster (lines 3–9). Mutations replaced methionyl residues with alanyl. The three positions were mutated simultaneously (scanc2/AAA), in pairs (scanc2/AAM, scanc2/AMA, scanc2/MAA), or singly (scanc2/AMM, scanc2/MAM, scanc2/MMA) under the control of ScANC2 regulatory sequences. A, yeast transformants were isolated and inoculated in complete, tryptophan-free liquid minimal medium containing 2% glucose as carbon source (YNB Glc W⁻). When cultures reached the log phase, cells were diluted to obtain 2 ×10⁵ to 2 ×10³ cells per 4 μl before spotting onto YNB Glc W⁻ (glucose) or rich lactate-containing medium (YPL) plates. Plates were incubated for 3 days (YNB Glc W⁻) or between 3 and 7 days (YPL) at 28 °C. B, shown are growth curves for strains ScANC2, scanc2/MAM, and scanc2/AMM in rich liquid medium (YPL). Cultures were inoculated with cells previously grown on YNB Glc W⁻ medium to obtain an initial A_{600 nm} = 0.1. Growth was monitored by measuring absorbance at 600 nm (A_{600 nm}) for ∼45, 65, and 85 h, depending on the yeast strain. Except for scanc2/AMM, which was a revertant strain, values are the means of at least three independent experiments.