Figure 5.

Test of HSP70A promoter deletion constructs for induction by MgPROTO. Two 5′truncations into the upstream region of the HSP70A promoter (Δ-209 and Δ-138) were converted into reporter genes by insertion of a 299 bp DNA fragment (TAG) into the 3′untranslated region of HSP70A (21) and co-transformed with a plasmid containing the ARG7 gene into an arg7 mutant. After an incubation for 20 h in the dark samples for RNA isolation were taken from subcultures that either had received MgPROTO (4 μM final concentration) for 1 h, were shifted from dark to light for 1 h (DLS), were shifted from 23°C to 40°C for 30 min (HS), or were incubated in the dark for another hour (CD). Ten micrograms of total RNA (2 μg for heat shock) was hybridized with the TAG probe, thereby specifically detecting the mRNA encoded by the promoter deletion constructs, or an HSP70A probe that hybridized to both HSP70A mRNAs.