CLONING OF THE DIHYDROXYACID DEHYDRATASE-ENCODING GENE (ILV3) FROM SACCHAROMYCES-CEREVISIAE

The biosynthesis of branched-chain amino acids (aa) involves three sha red pathways through which pyruvate or alpha-ketobutyrate are converte d into alpha-keto acids, precursors of valine, leucine or isoleucine. In eukaryotes, few of these common enzymes have been purified to homog eneity, and the whole complement of biosynthetic genes has not been cl oned from a single species. In yeasts, most of these genes (ILV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways. We have isolated Saccizaromyces cerevisiae genomic se quences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding g enes. The cloned sequences have been located to S. cerevisiae chromoso me X, mapped within 0.4 centiMorgans (cM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains. Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ILV3 gene, which codes for the yeast DAD. With our cloning of ILV3, yeast become s the only eukaryotic system from which all ILV genes have been cloned , thus allowing direct molecular analyses of their regulation.