MOLECULAR-CLONING AND NUCLEOTIDE SEQUENCING OF THE GAMMA-GLUTAMYLCYSTEINE SYNTHETASE GENE OF THE FISSION YEAST SCHIZOSACCHAROMYCES-POMBE

A DNA fragment encoding gamma-glutamylcysteine synthetase [EC 6.3.2.2] of Schizosaccharomyces pombe was cloned by complementation of the cad mium hypersensitivity of a S. pombe mutant deficient in the enzyme. Se quence analysis of the cloned DNA revealed that the enzyme was consist ed of 669 amino acid residues and was homologous to the enzymes of hum an liver, rat kidney, and Saccharomyces cerevisiae. The deduced amino acid sequence coincides with the amino acid sequences of the proteolyt ic peptides obtained from the purified enzyme. A cysteine residue was deduced to be important for catalytic activity by comparing the amino acid sequences of the enzymes of the four species, The gene contains o ne intron and the splicing point was confirmed by sequencing a cDNA am plified by PCR. Northern blot analysis showed an RNA of 2,200 bases hy bridized with the cloned gene.