Molecular characterisation of a male-specific serine/threonine phosphatase from Oesophagostomum dentatum (Nematoda: Strongylida), and functional analysis of homologues in Caenorhabditis elegans q Peter R. Boag a,c,1 , Peifeng Ren b , Susan E. Newton a , Robin B. Gasser c, * a Victorian Institute of Animal Science, Attwood, Victoria 3049, Australia b Syngenta Biotechnology Incorporated, North Carolina, USA c Department of Veterinary Science, The University of Melbourne, Werribee, Victoria 3030, Australia Received 23 September 2002; received in revised form 20 November 2002; accepted 3 December 2002 This paper is dedicated to the late Peter Nansen, a great parasitologist, philosopher and friend Abstract A male-specifically expressed sequence tag was used as a probe to screen adult male Oesophagostomum dentatum (Nematoda; Strongylida) gene libraries. The cDNA clones isolated coded for a serine/threonine protein phosphatase with , 85% identity to two Caenorhabditis elegans proteins implicated in reproduction. However, the genomic structures for the two species were distinct, in that the O. dentatum gene contained seven introns, whereas the C. elegans homologues contained three (two of which were conserved between the two nematodes). The promoters of all three nematode genes contained two putative GATA motifs separated by six to seven nucleotides and located within 100 nucleotides of the predicted transcriptional start site. RNA interference (RNAi) experiments in C. elegans, targetting the two homologues, revealed a consistent reduction in the number of progeny produced by treated worms, indicating a functional role in reproduction. Expression of green fluorescent protein, directed by the putative promoters for the C. elegans phosphatase genes, was analysed in transgenic C. elegans. The present results suggest that there is a significant degree of conservation between O. dentatum and C. elegans in the features and function of the serine/threonine protein phosphatase characterised, which should have implications for detailed investigations into molecular reproductive processes of some parasitic nematodes. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Oesophagostomum dentatum; Caenorhabditis elegans; Male-specific gene expression; Protein phosphatase; Double-stranded RNA interference 1. Introduction Protein phosphorylation/dephosphorylation represents a fundamental mechanism of post-transcriptional regulation of protein function. Protein phosphatases catalyse the removal of phosphate groups from specific residues of proteins (Barford, 1996) and can be divided into two main functional groups, namely the tyrosine phosphatases, which are usually membrane bound and are often involved in receptor-mediated signal transduction (Sarmiento et al., 1998), and the serine/threonine phosphatases, which are usually located in the cytoplasm of the cell and are often involved in signal transduction and transcriptional acti- vation (Barford et al., 1998). The protein phosphatase family can be further sub-divided into four classes (PP1, PP2A, PP2B and PP2C) based on substrate and inhibitor specificities (Barford et al., 1998). Classes PP1 and PP2A represent haloenzymes, which require the catalytic protein to be complexed with regulatory proteins involved in targetting and the regulation of phosphatase activity (Barford et al., 1998). Some members of class PP1 have been implicated in reproductive processes in a range of animals. For example, these serine/threonine phosphatases are involved in spermatogenesis and the regulation of sperm motility (Smith et al., 1996; Varmuza et al., 1999). 0020-7519/03/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0020-7519(02)00263-1 International Journal for Parasitology 33 (2003) 313–325 www.parasitology-online.com q Note: The nucleotide sequences reported in this paper are available in the EMBL, GenBank and DDJB databases under the Accession nos. AF496634 and AF496635. 1 Present address: Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, MA, USA. * Corresponding author. Tel.: þ 61-3-9731-2283; fax: þ61-3-9731-2366. E-mail address: robinbg@unimelb.edu.au (R.B. Gasser).