Molecular Organization of the Glutathione Reductase Gene inDrosophila melanogaster

Glutathione reductase catalyzes the conversion of the oxidized form of glutathione to regenerate reduced glutathione, which acts as a versatile intracellular reductant. The present study provides initial characterization of the glutathione reductase gene inDrosophila melanogasterand its response to experimentally induced oxidative stress.DrosophilacDNA clones were isolated, based on cross-hybridization to theMusca domesticaglutathione reductase cDNA. Genomic clones were isolated by cross-hybridization with theDrosophilacDNA as hybridization probe. Northern analysis of adultDrosophilapoly(A)+RNA, utilizing theDrosophilacDNA probe, revealed a hybridization signal in the 2-kb range. The entire sequence of one cDNA was determined. In addition to a coding domain of 1431 bases, the sequence included 206 bases upstream of a putative start codon and 355 bases downstream of a putative stop codon. Based on the cDNA sequence, the 476 amino acid sequence of theDrosophilaglutathione 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 glutathione reductase gene consists of at least two exons spanning approximately 5 kb. A first exon contains sequence for only the first 5 amino 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. TheDrosophilaglutathione reductase is single copy and its cytogenetic position, as determined byin situhybridization, 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 than threefold. Exposure of flies to hyperoxia also induced a 60% increase in the activity of glutathione reductase and augmented the concentration of total glutathione by ca. 40% following an initial drop. The present study, besides providing an initial molecular characterization of the glutathione reductase gene inDrosophila,demonstrates its dynamic involvement in response to experimentally induced oxidative stress.