MOLECULAR-ORGANIZATION OF THE GLUTATHIONE-REDUCTASE GENE IN DROSOPHILA-MELANOGASTER

Glutathione reductase catalyzes the conversion of the oxidized form of glutathione to regenerate reduced glutathione, which acts as a versat ile intracellular reductant. The present study provides initial charac terization of the glutathione reductase gene in Drosophila melanogaste r and its response to experimentally induced oxidative stress. Drosoph ila cDNA clones were isolated, based on cross-hybridization to the Mus ca domestica glutathione reductase cDNA. Genomic clones were isolated by cross-hybridization with the Drosophila cDNA as hybridization probe . Northern analysis of adult Drosophila poly(A)(+) RNA, utilizing the Drosophila cDNA probe, revealed a hybridization signal in the 2-kb ran ge. The entire sequence of one cDNA was determined. In addition to a c oding domain of 1431 bases, the sequence included 206 bases upstream o f a putative start codon and 355 bases downstream of a putative stop c odon. Based on the cDNA sequence, the 476 amino acid sequence of the D rosophila glutathione reductase gene was deduced and was found to have extensive similarities with the glutathione reductase gene from other species. Gene mapping of a 13-kb genomic fragment revealed that the g lutathione reductase gene consists of at least two exons spanning appr oximately 5 kb. A first exon contains sequence for only the first 5 am ino acids and the first base of the sixth and appears to be separated by a ca. 2.5-kb intron from the remainder of the coding region, which is confined to <2 kb. The Drosophila glutathione reductase is single c opy and its cytogenetic position, as determined by in situ hybridizati on, is 7D-E on the X chromosome. mRNA levels of glutathione reductase, measured by RT-PCR, increased in response to exposure to 100% ambient oxygen by almost twofold and administration of paraquat by greater th an threefold. Exposure of flies to hyperoxia also induced a 60% increa se in the activity of glutathione reductase and augmented the concentr ation of total glutathione by ca. 40% following an initial drop. The p resent study, besides providing an initial molecular characterization of the glutathione reductase gene in Drosophila, demonstrates its dyna mic involvement in response to experimentally induced oxidative stress . (C) 1997 Academic Press.