Isolation and partial characterization of the ARO1 gene of Saccharomyces cerevisiae

The AR01 cluster-gene of Saccharomyces cerevisiae is thought to be a single transcriptional unit encoding one polypeptide and five sequential enzymatic activities involved in the biosynthesis of the aromatic amino acids. The AR01 cluster-gene was previously isolated by complementation in Saccharomyces cerevisiae following transformation with a comprehensive yeast DNA library of BamHI restriction fragments inserted into the shuttle vector YEpl3. The original insert containing AR01 was 17.2 Kb BamHI fragment which complemented both nonsense and missense alleles of AR01 in yeast. In this study, subcloning of the 17.2 Kb AR01 fragment into pBR322, using Sau3AI partial digestion, further located the AR01 segment to a 4 Kb to 6 Kb region. Subsequent restriction digest mapping and subcloning of the AR01 6.5 Kb fragment with BamHI linkers led to a family of increasingly larger subclones from a common BamHI end: 1.1 Kb, 2.0 Kb, 2.9 Kb, 3.5 Kb, 4.3 Kb, 5.4 Kb, and 6.0 Kb. By using this family of subclones and a yeast AR01 nonsense mutant, complementation analysis demonstrated that the AR01 structural gene was located within the 3.5 Kb fragment. The 3.5 Kb fragment also complemented aroA, aroB, aroD, and aroE mutants of E. coli. Preliminary DNA sequencing studies and RNA/DNA hybridization studies have indicated the direction of transcription along the gene fragment.