Abstract
The PIS1 gene is required for de novo synthesis of phosphatidylinositol (PI), an essential phospholipid in Saccharomyces cerevisiae. PIS1 gene expression is unusual because it is uncoupled from the other phospholipid biosynthetic genes, which are regulated in response to inositol and choline. Relatively little is known about regulation of transcription of the PIS1 gene. We reported previously that PIS1 transcription is sensitive to carbon source. To further our understanding of the regulation of PIS1 transcription, we carried out a promoter deletion analysis that identified three regions required for PIS1 gene expression (upstream activating sequence (UAS) elements 1-3). Deletion of either UAS1 or UAS2 resulted in an approximately 45% reduction in expression, whereas removal of UAS3 yielded an 84% decrease in expression. A comparison of promoters among several Saccharomyces species shows that these sequences are highly conserved. Curiously, the UAS3 element region (-149 to -138) includes a Rox1p binding site. Rox1p is a repressor of hypoxic genes under aerobic growth conditions. Consistent with this, we have found that expression of a PIS1-cat reporter was repressed under aerobic conditions, and this repression was dependent on both Rox1p and its binding site. Furthermore, PI levels were elevated under anaerobic conditions. This is the first evidence that PI levels are affected by regulation of PIS1 transcription.
Highlights
Tion [9, 10], mRNA export from the nucleus [11,12,13,14,15], and vesicle trafficking [16]
PIS1 gene expression is unusual because it is uncoupled from the other phospholipid biosynthetic genes, which are regulated in response to inositol and choline
To further our understanding of the regulation of PIS1 transcription, we carried out a promoter deletion analysis that identified three regions required for PIS1 gene expression (upstream activating sequence (UAS) elements 1–3)
Summary
Media, and Growth Conditions—The S. cerevisiae strains used in this study were BRS1001 (MATa ade his leu112 can100 ura trp1–1), FY23 (MATa ura trp1⌬63 leu2⌬1) [44], and FY23⌬rox (MATa ura trp1⌬63 leu2⌬1 rox1::LEU2) [45]. Plasmid Construction—A nested set of PIS1 promoter deletions fused to the cat reporter gene was created by PCR using appropriate oligonucleotides (Table I). Fragments from appropriate pGEM-T derivatives were combined into pBM2015 to create PIS1 promoter internal deletions. Plasmid pANB1 was used as a control for Rox1p binding in electrophoretic mobility shift assay (EMSA) experiments. This pGEM-T derivative was constructed by cloning 150 bp of the ANB1 promoter Ϫ321 to Ϫ171 amplified by using primers ANB1 Ϫ321 and Ϫ171. Plasmid pPIS1-bend was generated for use in a circular permutation assay This plasmid was constructed by cloning a tandem direct repeat of PIS1 promoter sequences from Ϫ247 to Ϫ39 using primers PIS1 Ϫ247 and Ϫ39. Phospholipids were visualized with a PhosphorImager (Amersham Biosciences) and quantified using Imagequant software (Amersham Biosciences)
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