Complete sequence of a eukaryotic regulatory gene

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The pyrimidine pathway is essential for cell growth, with dihydroorotase as a key enzyme.
• In Saccharomyces cerevisiae, the URA4 gene encodes dihydroorotase and is transcriptionally regulated.
• Ureidosuccinic acid accumulation induces URA4 expression.

Purpose of the Study:

• To characterize the regulatory gene PPR2 involved in URA4 transcriptional control.
• To elucidate the molecular mechanism of PPR2 action in pyrimidine biosynthesis regulation.

Main Methods:

• Gene cloning and sequencing of PPR2 and its uninducible allele.
• Analysis of the PPR2 open reading frame and predicted protein structure.
• Mutation analysis using ochre suppressors.

Main Results:

• The PPR2 gene was cloned and sequenced, revealing a 384-nucleotide open reading frame potentially coding for a basic, oligomeric protein.
• An uninducible PPR2 allele contained an A-to-T transversion, creating an ochre codon and disrupting protein synthesis.
• Ochre suppressor mutations rescued the uninducible phenotype, confirming PPR2's role in URA4 transcription.

Conclusions:

• PPR2 encodes a regulatory protein essential for URA4 gene induction.
• The protein likely functions by directly binding to DNA to regulate URA4 transcription.
• Disruption of PPR2 function leads to an uninducible pyrimidine pathway.