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 cadmium hypersensitivity of a S. pombe mutant deficient in the enzyme. Sequence analysis of the cloned DNA revealed that the enzyme was consisted of 669 amino acid residues and was homologous to the enzymes of human liver, rat kidney, and Saccharomyces cerevisiae. The deduced amino acid sequence coincides with the amino acid sequences of the proteolytic 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 one intron and the splicing point was confirmed by sequencing a cDNA amplified by PCR. Northern blot analysis showed an RNA of 2,200 bases hybridized with the cloned gene.
Mutoh
N
N
Department of Genetics, Institute for Developmental Research, Aichi.
Nakagawa
C W
CW
Hayashi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D005800:Genes, Fungal; D015698:Genomic Library; D005721:Glutamate-Cysteine Ligase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004271:DNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005721:Glutamate-Cysteine Ligase