CIS - AND TRANS -ACTING REGULATION OF THE INO1 GENE OF SACCHAROMYCES CEREVISIAE

Abstract

The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate (I-1-P) synthase, which catalyzes the first committed step in the synthesis of the inositol-containing phospholipids. The activity and abundance of I-1-P synthase had previously been shown to be regulated by factors that affect phospholipid synthesis. This study makes use of the cloned INO1 gene to study both cis - and trans -acting factors involved in this regulation.;I-1-P synthase was shown to be encoded by a 1.8 kb poly(A){dollar}\sp+{dollar} RNA. The abundance of INO1 mRNA decreased when cells were grown in the presence of inositol, and decreased further when cells were grown in the presence of inositol and choline together, though choline alone had no effect. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of I-1-P synthase and results in excretion of inositol. The pattern of INO1 mRNA expression in cells containing a mutation affecting the synthesis of phosphatidylcholine (cho2) suggested that there is a regulatory link between the synthesis of the inositol- and choline-containing lipids.;A series of deletions from the 5{dollar}\sp\prime{dollar} end of the INO1 promoter were generated to identify the sequences involved in its expression. These constructions were integrated into the yeast genome and their expression was quantitated by S1 analysis. Sequences upstream of nucleotide {dollar}\sp-86{dollar}, relative to the start site of transcription, are required for INO1 expression. Removal of a region between {dollar}\sp-332{dollar} and {dollar}\sp-213{dollar} resulted in a high constitutive level of expression, suggesting that this region contains a negative site that normally functions to repress transcription of the INO1 gene.