MOLECULAR-CLONING AND CHARACTERIZATION OF THE SCHIZOSACCHAROMYCES-POMBE HIS3 GENE FOR USE AS A SELECTABLE MARKER

A DNA fragment which carries the his3 gene of Schizosaccharomyes pombe has been isolated and characterized for use as a selectable marker in transformations. The his3 gene encodes the imidazole acetol phosphate transaminase enzyme (E.C.2.6.1.9), which is responsible for converting imidazole acetol-P to histidinol-P in step 8 of histidine biosynthesis. The nucleotide sequences of a 2196 bp gene fragment and a corresponding cDNA clone were determined. Three intron sequences punctuate the 1451 bp coding region which generates a predicted polypeptide of 384 amino acids with a molecular mass of 42736 daltons. Northern analysis of his3 mRNAs indicates that the transcript is approximately 1.6 kb in size. Steady-state levels are down-regulated by nitrogen limitation but are unaffected by histidine starvation. The deduced amino acid sequence was compared to the Saccharomyces cerevisiae HIS5, Escherichia coli HisC, and Salmonella typhimurium HisC proteins, all of which are imidazole acetol phosphate transaminases. The S. pombe his3 protein was 49.5% identical to the S. cerevisiae HIS5 protein and 21.5% identity was found when all four proteins were compared. The shuttle vector pBG1 was constructed by subcloning the smallest functional region of his3 and the S. pombe ars1 sequence into pUC18 for use in transformation of His3 - S. pombe strains. New S. pombe strains in which the his3 gene was deleted have also been constructed.