Candas M, Sohal R S, Radyuk S N, Klichko V I, Orr W C
Department of Biological Sciences, Southern Methodist University, Dallas, Texas, 75275, USA.
Arch Biochem Biophys. 1997 Mar 15;339(2):323-34. doi: 10.1006/abbi.1996.9872.
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 in Drosophila melanogaster and its response to experimentally induced oxidative stress. Drosophila cDNA clones were isolated, based on cross-hybridization to the Musca 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 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 the Drosophila 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 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. The Drosophila glutathione reductase is single copy and its cytogenetic position, as determined by in situ hybridization, 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 in Drosophila, demonstrates its dynamic involvement in response to experimentally induced oxidative stress.
谷胱甘肽还原酶催化谷胱甘肽氧化形式的转化，以再生还原型谷胱甘肽，还原型谷胱甘肽作为一种多功能的细胞内还原剂。本研究对黑腹果蝇中的谷胱甘肽还原酶基因及其对实验诱导的氧化应激的反应进行了初步表征。基于与家蝇谷胱甘肽还原酶cDNA的交叉杂交，分离出果蝇cDNA克隆。以果蝇cDNA为杂交探针，通过交叉杂交分离出基因组克隆。利用果蝇cDNA探针，对成年果蝇多聚腺苷酸加尾RNA进行Northern分析，发现在2 kb范围内有杂交信号。测定了一个cDNA的完整序列。除了1431个碱基的编码结构域外，该序列在假定的起始密码子上游包含206个碱基，在假定的终止密码子下游包含355个碱基。根据cDNA序列，推导了果蝇谷胱甘肽还原酶基因的476个氨基酸序列，发现其与其他物种的谷胱甘肽还原酶基因有广泛的相似性。对一个13 kb基因组片段的基因定位显示，谷胱甘肽还原酶基因至少由两个外显子组成，跨度约为5 kb。第一个外显子仅包含前5个氨基酸的序列和第6个氨基酸的第一个碱基，并且似乎与编码区的其余部分被一个约2.5 kb的内含子隔开，编码区其余部分局限于小于2 kb。果蝇谷胱甘肽还原酶是单拷贝的，通过原位杂交确定其细胞遗传学位置在X染色体的7D-E处。通过RT-PCR测量，谷胱甘肽还原酶的mRNA水平在暴露于100%环境氧气时增加了近两倍，在施用百草枯时增加了三倍以上。果蝇暴露于高氧环境还导致谷胱甘肽还原酶活性增加60%，并在最初下降后使总谷胱甘肽浓度增加约40%。本研究除了对果蝇中的谷胱甘肽还原酶基因进行初步分子表征外，还证明了其在对实验诱导的氧化应激反应中的动态参与。
