Molecular and Biochemical Parasitology 109 (2000) 101 – 110 Identification of a stress-responsive Onchocerca olulus glutathione S-transferase (O -GST-3) by RT-PCR differential display  Eva Liebau a, *, Marie-Luise Eschbach a , Wilson Tawe b , Alexandra Sommer a , Peter Fischer a , Rolf D. Walter a , Kimberly Henkle-Du ¨ hrsen c a Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Bernhard -Nocht -Str. 74, D-20359 Hamburg, Germany b Laboratory of Molecular Parasitology, New York Blood Center, 310 East 67th Street, New York, NY 10021, USA c Institute for Genetics, Heinrich -Heine -Uniersity, Uniersita ¨tsstrasse 1, D-40225 Du ¨sseldorf, Germany Received 4 February 2000; received in revised form 16 March 2000; accepted 16 March 2000 Abstract The effects of oxidative insult on gene transcript levels in the filarial nematode Onchocerca olulus 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 (O -GST-3) possesses only 14 and 21% sequence identity with the other O. olulu s GSTs (O -GST-1 and O -GST-2, respectively). Interestingly, O -GST-3 shares higher sequence identity with GSTs that are upregulated due to environmental stress. In order to confirm the specific upregulation of the O -GST-3 transcripts identified by differential display and to analyze the mRNA levels of the other O -GSTs (O -GST-1 and O -GST-2) under elevated stress conditions, a semi-quantitative polymerase chain reaction-enzyme-linked immunosorbent assay was performed. The O -GST-3 gene transcript level increased dramat- ically in response to xanthine-xanthine oxidase and to a lesser extent with paraquat and plumbagin. In contrast, O -GST-1 and O -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 O -GST-3 is a critical enzyme in the defense against oxidative stress. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Glutathione S-transferase (GST); Differential display (DD); Polymerase chain reaction (PCR) www.elsevier.com/locate/parasitology  Note: Nucleotide sequence data reported in this paper have been submitted to the EMBL data base with the accession number AF203814 * Corresponding author. Tel.: +49-40-42818415; fax: +49-40-42818418. E-mail address: liebau@bni.uni-hamburg.de (E. Liebau). 0166-6851/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII:S0166-6851(00)00232-2