Molecular cloning of Drosophila gamma-glutamylcysteine synthetase by functional complementation of a yeast mutant.
gamma-Glutamylcysteine synthetase (GCS) catalyses a critical, rate-limiting step in glutathione synthesis. In this study we describe the isolation and characterisation of a GCS cDNA (pDmGCS4.3. 3) from Drosophila melanogaster by functional complementation of a Saccharomyces cerevisiae gsh1 mutant. Expression of pDmGCS4.3.3 in the yeast mutant partially restored glutathione levels and conferred resistance to methylglyoxal. The pDmGCS4.3.3 cDNA was found to be approx. 4.6 kb in length, containing a 2 kb fragment encoding an open reading frame with a high degree of deduced amino acid sequence identity with previously reported GCS sequences. In situ hybridisation revealed that the Drosophila GCS gene maps to 7D6-9 on the X chromosome.
Saunders
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Department of Anatomy, University of Dundee, Dundee, UK. r.d.saunders@open.ac.uk
McLellan
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D000595:Amino Acid Sequence; D000818:Animals; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics*; D005816:Genetic Complementation Test; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D005978:Glutathione / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment
D018076:DNA, Complementary; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione