FIG. 1.
The effect of the M232V mutation. (A) Yeast carrying a HSE-lacZ reporter gene, the hsfJ-Δ disruption, and an episomal plasmid expressing either wild-type HSF or HSFM232V were grown at 25°C, then assayed for β-galactosidase activity either directly or after 1 h at 37°C. (B) Protease-deficient yeast expressing either wild-type HSF or HSFM232V were used to prepare whole cell extracts after growth at 25°C or at 25°C followed by a 30-min heat shock at 37°C. Extracts were then used for gel mobility shift DNA binding assay using a 32P-labeled DNA probe (HSE4T) to which a single HSF trimer can bind. (C) The extracts from (B) were reanalyzed using a DNA probe (HSE6T) to which two trimers can bind simultaneously. The 25°C sample of wild-type was also assayed with DNA probe 4T, in order to identify the position of the DNA-bound trimer.