N. Mutoh et al., MOLECULAR-CLONING AND NUCLEOTIDE SEQUENCING OF THE GAMMA-GLUTAMYLCYSTEINE SYNTHETASE GENE OF THE FISSION YEAST SCHIZOSACCHAROMYCES-POMBE, Journal of Biochemistry, 117(2), 1995, pp. 283-288
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.