Liebau E, Eschbach M L, Tawe W, Sommer A, Fischer P, Walter R D, Henkle-Dührsen K
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Mol Biochem Parasitol. 2000 Jul;109(2):101-10. doi: 10.1016/s0166-6851(00)00232-2.
The effects of oxidative insult on gene transcript levels in the filarial nematode Onchocerca volvulus were investigated using differential display RT-PCR. Oxidative stress was applied with the reagents paraquat, plumbagin and xanthine-xanthine oxidase. In all three cases, a cDNA fragment encoding a novel glutathione S-transferase (GST) resembling members of the theta-class was identified as upregulated (PQ29, PG112, XOD26). The subsequently isolated full-length cDNA harbors a 753-bp open reading frame encoding a GST with 268 amino acid residues and a predicted molecular mass of 31 kDa. This stress-responsive GST (Ov-GST-3) possesses only 14 and 21% sequence identity with the other O. volvulus GSTs (Ov-GST-1 and Ov-GST-2, respectively). Interestingly, Ov-GST-3 shares higher sequence identity with GSTs that are upregulated due to environmental stress. In order to confirm the specific upregulation of the Ov-GST-3 transcripts identified by differential display and to analyze the mRNA levels of the other Ov-GSTs (Ov-GST-1 and Ov-GST-2) under elevated stress conditions, a semi-quantitative polymerase chain reaction-enzyme-linked immunosorbent assay was performed. The Ov-GST-3 gene transcript level increased dramatically in response to xanthine-xanthine oxidase and to a lesser extent with paraquat and plumbagin. In contrast, Ov-GST-1 and Ov-GST-2 did not show any significant alterations in their steady-state mRNA levels in response to oxidative stress when examining the same mRNA samples. The present study clearly demonstrates that Ov-GST-3 is a critical enzyme in the defense against oxidative stress.
利用差异显示逆转录聚合酶链反应（RT-PCR）研究了氧化损伤对丝状线虫旋盘尾丝虫基因转录水平的影响。使用百草枯、白花丹素和黄嘌呤-黄嘌呤氧化酶试剂施加氧化应激。在所有这三种情况下，一个编码类似于θ类成员的新型谷胱甘肽S-转移酶（GST）的cDNA片段被鉴定为上调（PQ29、PG112、XOD26）。随后分离出的全长cDNA包含一个753 bp的开放阅读框，编码一个具有268个氨基酸残基、预测分子量为31 kDa的GST。这种应激反应性GST（Ov-GST-3）与其他旋盘尾丝虫GST（分别为Ov-GST-1和Ov-GST-2）的序列同一性仅为14%和21%。有趣的是，Ov-GST-3与因环境应激而上调的GST具有更高的序列同一性。为了证实差异显示所鉴定的Ov-GST-3转录本的特异性上调，并分析其他Ov-GST（Ov-GST-1和Ov-GST-2）在应激条件升高时的mRNA水平，进行了半定量聚合酶链反应-酶联免疫吸附测定。Ov-GST-3基因转录水平在黄嘌呤-黄嘌呤氧化酶作用下显著增加，在百草枯和白花丹素作用下增加程度较小。相比之下，当检测相同的mRNA样本时，Ov-GST-1和Ov-GST-2在氧化应激下其稳态mRNA水平未显示任何显著变化。本研究清楚地表明，Ov-GST-3是抵御氧化应激的关键酶。
