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Molecular cloning of Drosophila γ‐glutamylcysteine synthetase by functional complementation of a yeast mutant
Author(s) -
Saunders Robert D.C.,
McLellan Lesley I.
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01148-0
Subject(s) - complementation , mutant , biology , complementary dna , microbiology and biotechnology , saccharomyces cerevisiae , drosophila melanogaster , open reading frame , molecular cloning , protein fragment complementation assay , gene , genetics , yeast , cloning (programming) , biochemistry , peptide sequence , computer science , programming language
γ‐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.