FIG 9.

Plasmid-borne Asp1^365–920 expression rescues the temperature-sensitive nongrowth phenotype of the cut7-466 strain. (A) Serial dilution patch tests (10⁴ to 10¹ cells) of the cut7-466 strain transformed with vector (control), asp1^1–364, or asp1^365–920 plasmid. Transformants were grown in thiamine-free medium under plasmid selective conditions at the indicated temperatures for 6 days. (B) Diagrammatic illustration of the survival of transformed cut7-466 cells grown at the restrictive temperature of 28°C. Transformants were pregrown at 25°C under plasmid selective conditions that allowed expression of the asp1 variants before plating cells at 28°C. Colony formation was assayed for 1,000 cells/transformant. ***, P < 0.005 (χ² test). (C and D) Serial dilution patch tests (10⁴ to 10¹ cells) of the indicated strains transformed with the same plasmids as for panel A. The absence of thiamine results in derepression of the nmt1⁺ promoter, which controls asp1^1–364 and asp1^365–920 expression. Plates were incubated at 25°C for 5 days.