FIG. 5.

S1 nuclease digestion assay of INO4 mRNA. A single-stranded PCR probe was hybridized to total cellular mRNA from the wild type (WT) strain (BRS1001) transformed with pJA201, and an ino4Δ mutant strain (BRS2004). BRS1001 was grown in leucine-lacking synthetic medium containing 75 μM inositol and 1 mM choline, while BRS2004 was grown in complete synthetic medium containing 75 μM inositol and 1 mM choline. S1 digestion fragments were run on a denaturing polyacrylamide gel next to the INO4 sequence that was generated with the same primer used to synthesize the probe. Different exposures of the sequencing ladder (1 day) and S1 digestion products (4 days) are shown. Nucleotide assignment of INO4 mRNA start sites. The stippled box represents the major start site. The solid boxes represent minor start sites. Sequences refer to the exact nucleotides present in the respective promoter deletions.